Methods Related to Wound Healing

ABSTRACT

The invention is directed to methods for the treatment of wounds. Such methods utilize novel compositions, including but not limited to amnion-derived multipotent cells (herein referred to as AMP cells), conditioned media derived therefrom (herein referred to as amnion-derived cellular cytokine suspension or ACCS), cell lysates derived therefrom, cell products derived therefrom, each alone or in combination.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to U.S.Provisional Application No. 60/666,949, filed Mar. 31, 2005, U.S.Provisional Application No. 60/699,257, filed Jul. 14, 2005, U.S.Provisional Application No. 60/742,067, filed Dec. 2, 2005, and under 35USC §120 to U.S. Utility application Ser. No. 11/333,849, filed Jan. 18,2006 (now abandoned), and U.S. Utility application Ser. No. 11/392,892,filed Mar. 29, 2006, the contents of which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with United States government supportawarded by the following agency: U.S. Army Medical Research AcquisitionActivity, ERMS #06100002 and #06153010. The United States may havecertain rights to this invention.

FIELD OF THE INVENTION

The field of the invention is directed to methods for the treatment ofwounds. Such methods utilize novel compositions, including but notlimited to extraembryonic cytokine secreting cells (herein referred toas ECS cells), including, but not limited to, amnion-derived multipotentcells (herein referred to as AMP cells), conditioned media derivedtherefrom (herein referred to as amnion-derived cellular cytokinesuspension or ACCS), cell lysates derived therefrom, and cell productsderived therefrom, each alone or in combination.

DESCRIPTION OF RELATED ART

Amniotic membranes have been used clinically as wound dressing for burnpatients for over 100 years to promote epithelialization, reduce pain,and prevent infection (Robson, M. C. and Krizek, T. J. (1973) Ann ofSurg, 177:144-149; Robson, M. C., et al., (1973) Jour Surg Res14:431-434; Robson, M. C., et al., (1973) Surgery, Ob & Gyn,136:904-906; Robson, M. C. and Krizek, T. J., (1974) Connecticut Med38:449-451; Krizek, T. J. and Robson, M. C., A rebirth of amnioticmembranes, in Marchac (Ed): Transactions of VI International Congress ofPlastic and Reconstructive Surgery, Masson, Inc., NY, 1976; Kucan, J.O., et al, (1982) Ann Plast Surg 8:523-527; Wu, C-H, et al., (2003)British J Dermatol 148:236-245; Bose, B. (1979) Ann R Coll Surg Engl,61:444-7; Sawhney, C. P. (1989) Burns, 15:339-42, Thomson, P. D., Parks,D. H. (1981) Ann Plast Surg, 7:354-6). US2003/0235580 describes a methodof delivering therapeutic molecules to skin using amniotic epithelialcells. US2004/0057938 describes the use of a human amniotic membranecomposition for prophylaxis and treatment of diseases and conditions ofthe eye and skin. U.S. Pat. No. 4,361,552 describes a method of treatinga wound or burn, which comprises covering the surface of the wound orburn with a cross-linked amnion dressing. US2004/0170615 describes theuse of compounds expressed in fetal tissue for use in skin repair andthe improvement of skin appearance.

BACKGROUND OF THE INVENTION

Placental tissue is abundantly available as a discarded source of manypotentially useful cell types including a type of multipotent cellcalled placental-derived cells. Although discarded at parturition aspart of the placental membranes, lineage analysis shows that, theepithelial layer of the amnion, from which such multipotent cells can beisolated, is uniquely descended from the epiblast in embryonicdevelopment. The epiblast contains the cells that will ultimatelydifferentiate into the embryo and cells that will give rise to anextraembryonic tissue, the amnion. Thus far, only four cell types havebeen described in the literature as being pluripotent. These are theinner cell mass (ICM) of the pre-implantation embryo, which gives riseto the epiblast, the epiblast itself, embryonic stem (ES) and embryonicgerm cells (EG). Thus, identification, purification and propagation of amultipotent cell population from discarded amnion tissue would providean extremely valuable source of stem cells for replacement cell therapy.

With an average yield of over 200 million cells per placenta, largenumbers of cells are available from this source. If these cells were tobecome useful cells for transplantation medicine, they could provide anearly inexhaustible supply of starting material in every part of theworld. No stem cell source provides such a large starting population ofcells, and collection does not require an invasive or destructiveprocedure. Furthermore, there are no ethical, religious or social issuesassociated with these cells as the tissue is derived from the placenta.

Another important consideration in stem cell therapies is immunetolerance. In humans, the protein expression of the cell surface markerHLA-G was originally thought to be restricted to immune-privileged sitessuch as placenta, as well as related cells, including some isolated fromamniotic fluid, placental macrophages, and cord blood, thus implicatingits role in maternal-fetal tolerance (Urosevic, M. and Dummer, R. (2002)ASHI Quarterly; 3rd Quarter 2002:106-109). Additionally, studiesinvolving heart-graft acceptance have suggested that the proteinexpression of HLA-G may enhance graft tolerance (Lila, N., et al. (2000)Lancet 355:2138; Lila, N. et al. (2002) Circulation 105:1949-1954).HLA-G protein is not expressed on the surface of undifferentiated ordifferentiated embryonic stem cells (Drukker, M, et al. (2002) PNAS99(15):9864-9869). Thus, it is desirable that stems cells intended forcell-based therapies express HLA-G protein.

Placental-derived cells have been shown to secrete many cytokines andgrowth factors including prostaglandin E2, PGES, TGF-β, EGF, IL-4, IL-8,TNF, interferons, activin A, noggin, bFGF, some neuroprotective factors,and many angiogenic factors (Koyano et al., (2002) Develop. GrowthDiffer. 44:103-112; Blumenstein et al. (2000) Placenta 21:210-217;Tahara et al. (1995) J. Clin. Endocrinol. Metabol. 80:138-146;Paradowska et al. (1997) Placenta 12:441-446; Denison et al. (1998) Hum.Reprod. 13:3560-3565; Keelan et al. (1998) Placenta 19:429-434; Uchidaet al. (2000) J. Neurosci. Res. 62:585-590; Sun et al. (2003) J. Clin.Endocrinol. Metabol. 88(11):5564-5571; Marvin et al. (2002) Am. J.Obstet. Gynecol. 187(3):728-734). Many of these cytokines are associatedwith wound healing and some have been credited with contributing toscarless healing in the fetus. Fetal skin has much more effective repairmechanisms than adult skin and, once wounded, it is able to heal withoutthe formation of scars. This capability does appear to require the fetalimmune system, fetal serum, or amniotic fluid (Bleacher J C, et al., JPediatr Surg 28: 1312-4, 1993); Ihara S, Motobayashi Y., Development114: 573-82. 1992). Such abilities of fetal tissue have led to thesuggested use of compounds produced by fetal tissue for regeneratingand/or improving the appearance of skin (see, for example, US2004/0170615, which is incorporated by reference in its entiretyherein).

Approximately 50 million surgical procedures are performed in the UnitedStates each year. An additional 50 million wounds result from traumaticinjuries. Subsequent acute wound healing failure at any anatomic siteresults in increased morbidity and mortality. Non-limiting examples ofacute wound failure include muscle, fascial and skin dehiscence,incisional hernia formation, gastrointestinal fistulization and vascularanastamotic leaks. Besides the immediate functional disability, acutewounds that fail usually go on to form disabling scars.

Incisional hernias of the abdominal wall provide an excellent paradigmto study the mechanism and outcome of acute wound healing failure.Large, prospective, well-controlled studies have shown that 11-20% ofover 4 million abdominal wall fascial closures fail leading to ventralincisional hernia formation. Even after repair of acute wound failure,recurrence rates remain as high as 58%. Improvements in suture material,stitch interval, stitch distance from the margin of the wound, andadministration of prophylactic antibiotics to avoid infectionsignificantly decreased the rates of clinically obvious acute wounddehiscence, but only led to small decreases in the rates of ventralhernia formation and recurrence. The introduction of tissue prostheses,typically synthetic meshes, to create a tension-free bridge or patch ofthe myo-fascial defect reduced first recurrence rates significantly,supporting the concept that mechanical factors predominate in thepathogenesis of recurrent hernia.

Traditional surgical teaching is that laparotomy wound failure is a rareevent, with reported “fascial dehiscence” rates clustered around 0.1%.One prospective study found that the true rate of laparotomy woundfailure is closer to 11%, and that the majority of these (94%) go on toform incisional hernias during the first three years after abdominaloperations. This is more in line with the high incidence of incisionalhernia formation. The real laparotomy wound failure rate is therefore100 times what most surgeons think it is. In simplest terms, mostincisional hernias are derived from clinically occult laparotomy woundfailures, or occult fascial dehiscences. The overlying skin wound heals,concealing the underlying myofascial defect. This mechanism of earlymechanical laparotomy wound failure is more consistent with modern acutewound healing science.

BRIEF SUMMARY OF THE INVENTION

It is an object of the instant invention to provide novel methods forthe treatment of wounds. Such methods utilize novel compositionsincluding extraembryonic cytokine secreting cells (herein referred to asECS cells), conditioned media derived therefrom, cell lysates derivedtherefrom, and cell products derived therefrom, each alone and/or incombination with each other and/or with other agents including activeand/or inactive agents. In a particular preferred embodiment, themethods utilize novel compositions including, but not limited to,amnion-derived multipotent cells (herein referred to as AMP cells),conditioned media derived therefrom (herein referred to asamnion-derived cellular cytokine suspension or ACCS), cell lysatesderived therefrom, and cell products derived therefrom, each aloneand/or in combination with each other and/or with other agents includingactive and/or inactive agents. In a most preferred embodiment, woundsare treated with ACCS, including pooled ACCS.

It is also an object of the instant invention to promote acceleratedwound healing using the novel compositions described herein. It isfurther an object of the invention to reduce or prevent scarringfollowing wound healing; to promote the formation of stronger healedwounds by increasing tensile strength and/or breaking strength; and toprevent or reduce wound healing failure, in particular, herniaformation, using the novel compositions described herein.

Accordingly, a first aspect of the invention is a method for promotingaccelerated wound healing in a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of oneor more compositions comprising ECS cells, conditioned media derivedtherefrom, cell lysate derived therefrom or cell products derivedtherefrom. In one embodiment the ECS cells are AMP cells. In a preferredembodiment the AMP cells are pooled AMP cells. In another embodiment theconditioned media is ACCS. In a preferred embodiment the ACCS is pooledACCS. In yet another embodiment the cell lysates are AMP cell lysates.In a preferred embodiment the AMP cell lysates are pooled AMP celllysates. In yet another embodiment the cell products are AMP cellproducts. In a preferred embodiment the AMP cell products are pooled AMPcell products. In still another embodiment the patient is a humanpatient.

A second aspect of the invention is a method for decreasing woundfailure in a surgical patient in need thereof comprising administeringto the patient a therapeutically effective amount of one or morecompositions comprising ECS cells, conditioned media derived therefrom,cell lysate derived therefrom or cell products derived therefrom.

A third aspect of the invention is a method for increasing tensilestrength and breaking strength of a wound in a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of one or more compositions comprising ECS cells, conditionedmedia derived therefrom, cell lysate derived therefrom or cell productsderived therefrom.

In one embodiment of aspects one, two and three of the invention the ECScells are AMP cells. In a preferred embodiment the AMP cells are pooledAMP cells. In another embodiment the conditioned media is ACCS. In apreferred embodiment the ACCS is pooled ACCS. In yet another embodimentthe cell lysates are AMP cell lysates and in a preferred embodiment theAMP cell lysates are pooled AMP cell lysates. In yet another embodimentthe cell products are AMP cell products and in a preferred embodimentthe AMP cell products are pooled AMP cell products. In still anotherembodiment the patient is a human patient.

In other embodiments the wound is a congenital wound or an acquiredwound. In preferred embodiments the congenital wound is epidermolysis orscalp aplasia. In other preferred embodiments the acquired wound is anacute wound or a chronic wound. In still other preferred embodiments theacute wound is trauma or a surgical incision and the trauma wound is aburn, open fracture or avulsion. In still other preferred embodimentsthe chronic wound is a pressure ulcer, venous ulcer, diabetic ulcer,sickle cell ulcer or peptic ulcer.

A fourth aspect of the invention is a method to reduce the occurrence oftissue scarring and adhesion formation during the wound healing processafter surgery, which comprises applying to the surgical site atherapeutically effective amount of AMP cells and/or ACCS. In apreferred embodiment the AMP cells and ACCS are pooled AMP cells andpooled ACCS. In another preferred embodiment the applying of AMP cellsand/or ACCS to the surgical site occurs prior to the surgical procedure.In another preferred embodiment the applying of AMP cells and/or ACCS tothe surgical site occurs during the surgical procedure. In still anotherpreferred embodiment the applying of AMP cells and/or ACCS to thesurgical site occurs or after the surgical procedure. In other preferredembodiments, the applying of AMP cells and/or ACCS to the surgical siteoccurs prior to, during and after the surgical procedure. Othercombinations for applying of AMP cells and/or ACCS to the surgical siteare contemplated by the methods of the invention.

A fifth aspect of the invention is a method for stimulating growth orregeneration of epidermal cells and inhibiting fibrosis and collagencontraction in a patient in need thereof comprising contacting thepatient's epidermal cells with a therapeutic amount of AMP cells and/orACCS. In a preferred embodiment the AMP cells and ACCS are pooled AMPcells and pooled ACCS. In still another embodiment the patient is ahuman patient.

A sixth aspect of the invention is a method for preventing keloid and/orhypertrophic scar formation at the site of a wound in a patient in needthereof comprising contacting the patient's wound with an amount of AMPcells and/or ACCS sufficient to stimulate growth and regeneration ofepidermal cells and inhibit fibrosis and collagen contraction. In apreferred embodiment the AMP cells and ACCS are pooled AMP cells andpooled ACCS. In still another embodiment the patient is a human patient.

A seventh aspect of the invention is a cosmetic preparation comprisingone or more compositions comprising AMP cells, ACCS, AMP cell lysates orAMP cell products. In a preferred embodiment the AMP cells are pooledAMP cells. In a preferred embodiment the ACCS is pooled ACCS. In yetanother the AMP cell lysates are pooled AMP cell lysates. In yet anotherembodiment the AMP cell products are pooled AMP cell products.

Other features and advantages of the invention will be apparent from theaccompanying description, examples and the claims. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference. In case of conflict, the present specification, includingdefinitions, will control.

DEFINITIONS

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

As defined herein, a “gene” is the segment of DNA involved in producinga polypeptide chain; it includes regions preceding and following thecoding region, as well as intervening sequences (introns) betweenindividual coding segments (exons).

As used herein, the term “protein marker” means any protein moleculecharacteristic of the plasma membrane of a cell or in some cases of aspecific cell type.

As used herein, “enriched” means to selectively concentrate or toincrease the amount of one or more materials by elimination of theunwanted materials or selection and separation of desirable materialsfrom a mixture (i.e. separate cells with specific cell markers from aheterogeneous cell population in which not all cells in the populationexpress the marker).

As used herein, the term “substantially purified” means a population ofcells substantially homogeneous for a particular marker or combinationof markers. By substantially homogeneous is meant at least 90%, andpreferably 95% homogeneous for a particular marker or combination ofmarkers.

The term “placenta” as used herein means both preterm and term placenta.

As used herein, the term “totipotent cells” shall have the followingmeaning. In mammals, totipotent cells have the potential to become anycell type in the adult body; any cell type(s) of the extraembryonicmembranes (e.g., placenta). Totipotent cells are the fertilized egg andapproximately the first 4 cells produced by its cleavage.

As used herein, the term “pluripotent stem cells” shall have thefollowing meaning. Pluripotent stem cells are true stem cells with thepotential to make any differentiated cell in the body, but cannotcontribute to making the components of the extraembryonic membraneswhich are derived from the trophoblast. The amnion develops from theepiblast, not the trophoblast. Three types of pluripotent stem cellshave been confirmed to date: Embryonic Stem (ES) Cells (may also betotipotent in primates), Embryonic Germ (EG) Cells, and EmbryonicCarcinoma (EC) Cells. These EC cells can be isolated fromteratocarcinomas, a tumor that occasionally occurs in the gonad of afetus. Unlike the other two, they are usually aneuploid.

As used herein, the term “multipotent stem cells” are true stem cellsbut can only differentiate into a limited number of types. For example,the bone marrow contains multipotent stem cells that give rise to allthe cells of the blood but may not be able to differentiate into othercells types.

As used herein, the term “extraembryonic tissue” means tissue locatedoutside the embryonic body which is involved with the embryo'sprotection, nutrition, waste removal, etc. Extraembryonic tissue isdiscarded at birth. Extraembryonic tissue includes but is not limited tothe amnion, chorion (trophoblast and extraembryonic mesoderm includingumbilical cord and vessels), yolk sac, allantois and amniotic fluid(including all components contained therein). Extraembryonic tissue andcells derived therefrom have the same genotype as the developing embryo.

As used herein, the term “extraembryonic cytokine secreting cells” or“ECS cells” means a population of cells derived from the extraembryonictissue which have the characteristic of secreting a unique combinationof physiologically relevant cytokines in a physiologically relevanttemporal manner into the extracellular space or into surrounding culturemedia. In one embodiment, the ECS cells secrete at least one cytokineselected from VEGF, Angiogenin, PDGF and TGFβ2 and at least one MMPinhibitor selected from TIMP-1 and TIMP-2. In another embodiment, theECS cells secrete more than one cytokine selected from VEGF, Angiogenin,PDGF and TGFβ2 and more than one MMP inhibitor selected from TIMP-1 andTIMP-2. In a preferred embodiment, the ECS cells secrete the cytokinesVEGF, Angiogenin, PDGF and TGFβ2 and the MMP inhibitors TIMP-1 andTIMP-2. The physiological range of the cytokine or cytokines in theunique combination is as follows: ˜5-16 ng/ml for VEGF, ˜3.5-4.5 ng/mlfor Angiogenin, ˜100-165 pg/ml for PDGF, ˜2.5-2.7 ng/ml for TGFβ2, ˜0.68μg ml for TIMP-1 and ˜1.04 μg/ml for TIMP-2. ECS cells may be selectedfrom populations of cells and compositions described in this applicationand in US2003/0235563, US2004/0161419, US2005/0124003, U.S. ProvisionalApplication Nos. 60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S.application Ser. No. 11/333,849, U.S. application Ser. No. 11/392,892,PCTUS06/011392, US2006/0078993, PCT/US00/40052, U.S. Patent No.7,045,148, US2004/0048372, and US2003/0032179, the contents of which areincorporated herein by reference in their entirety.

As used herein, the term “amnion-derived multipotent progenitor cell” or“AMP cell” means a specific population of ECS cells that are epithelialcells derived from the amnion. In addition to the characteristicsdescribed above for ECS cells, AMP cells have the followingcharacteristics. They grow without feeder layers, do not express theprotein telomerase and are non-tumorigenic. AMP cells do not express thehematopoietic stem cell marker CD34 protein. The absence of CD34positive cells in this population indicates the isolates are notcontaminated with hematopoietic stem cells such as umbilical cord bloodor embryonic fibroblasts. Virtually 100% of the cells react withantibodies to low molecular weight cytokeratins, confirming theirepithelial nature. Freshly isolated AMP cells will not react withantibodies to the stem/progenitor cell markers c-kit (CD117) and Thy-1(CD90). Several procedures used to obtain cells from full term orpre-term placenta are known in the art (see, for example, US2004/0110287; Anker et al., 2005, Stem Cells 22:1338-1345; Ramkumar etal., 1995, Am. J. Ob. Gyn. 172:493-500). However, the methods usedherein provide improved compositions and populations of cells. AMP cellshave previously been described as “amnion-derived cells” (see U.S.Provisional Application Nos. 60/666,949, 60/699,257, 60/742,067, U.S.Provisional Application Nos. 60/813,759, U.S. application Ser. No.11/333,849, U.S. application Ser. No. 11/392,892, and PCTUS06/011392,each of which is incorporated herein in its entirety).

By the term “animal-free” when referring to compositions, growthconditions, culture media, etc. described herein, is meant that noanimal-derived materials, such as animal-derived serum, other than humanmaterials, such as native or recombinantly produced human proteins, areused in the preparation, growth, culturing, expansion, or formulation ofthe composition or process.

By the term “expanded”, in reference to cell compositions, means thatthe cell population constitutes a significantly higher concentration ofcells than is obtained using previous methods. For example, the level ofcells per gram of amniotic tissue in expanded compositions of AMP cellsis at least 50 and up to 150 fold higher than the number of cells in theprimary culture after 5 passages, as compared to about a 20 foldincrease in such cells using previous methods. In another example, thelevel of cells per gram of amniotic tissue in expanded compositions ofAMP cells is at least 30 and up to 100 fold higher than the number ofcells in the primary culture after 3 passages. Accordingly, an“expanded” population has at least a 2 fold, and up to a 10 fold,improvement in cell numbers per gram of amniotic tissue over previousmethods. The term “expanded” is meant to cover only those situations inwhich a person has intervened to elevate the number of the cells.

As used herein, the term “passage” means a cell culture technique inwhich cells growing in culture that have attained confluence or areclose to confluence in a tissue culture vessel are removed from thevessel, diluted with fresh culture media (i.e. diluted 1:5) and placedinto a new tissue culture vessel to allow for their continued growth andviability. For example, cells isolated from the amnion are referred toas primary cells. Such cells are expanded in culture by being grown inthe growth medium described herein. When such primary cells aresubcultured, each round of subculturing is referred to as a passage. Asused herein, “primary culture” means the freshly isolated cellpopulation.

As used herein, “conditioned medium” is a medium in which a specificcell or population of cells has been cultured, and then removed. Whencells are cultured in a medium, they may secrete cellular factors thatcan provide support to or affect the behavior of other cells. Suchfactors include, but are not limited to hormones, cytokines,extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines,receptors, inhibitors and granules. The medium containing the cellularfactors is the conditioned medium. Examples of methods of preparingconditioned media are described in U.S. Pat. No. 6,372,494 which isincorporated by reference in its entirety herein. As used herein,conditioned medium also refers to components, such as proteins, that arerecovered and/or purified from conditioned medium or from ECS cells,including AMP cells.

As used herein, the term “amnion-derived cellular cytokine suspension”or “ACCS” means conditioned medium that has been derived from AMP cellsor expanded AMP cells.

The term “physiological level” as used herein means the level that asubstance in a living system is found and that is relevant to the properfunctioning of a biochemical and/or biological process.

As used herein, the term “pooled” means a plurality of compositions thathave been combined to create a new composition having more constant orconsistent characteristics as compared to the non-pooled compositions.For example, pooled AMP cells have more constant or consistentcharacteristics compared to non-pooled AMP cells.

The term “therapeutically effective amount” means that amount of atherapeutic agent necessary to achieve a desired physiological effect(i.e. accelerated wound healing).

The term “lysate” as used herein refers to the composition obtained whencells, for example, AMP cells, are lysed and optionally the cellulardebris (e.g., cellular membranes) is removed. This may be achieved bymechanical means, by freezing and thawing, by sonication, by use ofdetergents, such as EDTA, or by enzymatic digestion using, for example,hyaluronidase, dispase, proteases, and nucleases.

As used herein, the term “substrate” means a defined coating on asurface that cells attach to, grown on, and/or migrate on. As usedherein, the term “matrix” means a substance that cells grow in or onthat may or may not be defined in its components. The matrix includesboth biological and non-biological substances. As used herein, the term“scaffold” means a three-dimensional (3D) structure (substrate and/ormatrix) that cells grow in or on. It may be composed of biologicalcomponents, synthetic components or a combination of both. Further, itmay be naturally constructed by cells or artificially constructed. Inaddition, the scaffold may contain components that have biologicalactivity under appropriate conditions.

As used herein, the term “pharmaceutically acceptable” means that thecomponents, in addition to the therapeutic agent, comprising theformulation, are suitable for administration to the patient beingtreated in accordance with the present invention.

As used herein, the term “tissue” refers to an aggregation of similarlyspecialized cells united in the performance of a particular function.

As used herein, the term “therapeutic protein” includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors.

As used herein “germ cells” means embryonic germ cells, adult germ cellsand the cells that they give rise to (i.e. oocyte and sperm).

The term “transplantation” as used herein refers to the administrationof a composition comprising cells that are either in anundifferentiated, partially differentiated, or fully differentiated forminto a human or other animal.

As used herein, the terms “a” or “an” means one or more; at least one.

As used herein, the term “adjunctive” means jointly, together with, inaddition to, in conjunction with, and the like.

As used herein, the term “co-administer” can include simultaneous orsequential administration of two or more agents.

“Treatment,” “treat,” or “treating,” as used herein covers any treatmentof a disease or condition of a mammal, particularly a human, andincludes: (a) preventing the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet been diagnosed as having it; (b) inhibiting the disease orcondition, i.e., arresting its development; (c) relieving and orameliorating the disease or condition, i.e., causing regression of thedisease or condition; or (d) curing the disease or condition, i.e.,stopping its development or progression. The population of subjectstreated by the methods of the invention includes subjects suffering fromthe undesirable condition or disease, as well as subjects at risk fordevelopment of the condition or disease.

As used herein, a “wound” is any disruption, from whatever cause, ofnormal anatomy (internal and/or external anatomy) including but notlimited to traumatic injuries such as mechanical, thermal, andincisional injuries; elective injuries such as surgery and resultantincisional hernias; acute wounds, chronic wounds, infected wounds, andsterile wounds, as well as wounds associated with disease states (i.e.ulcers caused by diabetic neuropathy). A wound is dynamic and theprocess of healing is a continuum requiring a series of integrated andinterrelated cellular processes that begin at the time of wounding andproceed beyond initial wound closure through arrival at a stable scar.These cellular processes are mediated or modulated by humoral substancesincluding but not limited to cytokines, lymphokines, growth factors, andhormones. In accordance with the subject invention, “wound healing”refers to improving, by some form of intervention, the natural cellularprocesses and humoral substances such that healing is faster, and/or theresulting healed area has less scaring and/or the wounded area possessestissue tensile strength that is closer to that of uninjured tissue.

As used herein, the term “cosmeceutical” means cosmetic products thatmay have drug-like benefits. Examples of products typically labeled ascosmeceuticals include anti-aging creams and moisturizers.Cosmeceuticals may contain potentially active ingredients such asvitamins, phytochemicals, enzymes, antioxidants, and essential oils.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized CellsAnd Enzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Therapeutic Uses

Wound Healing

The instant invention is based upon the discovery that undifferentiated,partially differentiated or fully differentiated ECS cells, and inparticular, AMP cells, ACCS derived therefrom, cell lysates therefrom,cell products derived therefrom, and extracellular matrices therefrom,alone or in combination with each other and/or other agents, includingactive and non-active agents, as well as compositions of ECS cells asdefined herein, can accelerate the wound healing process for all woundtypes, particularly when administered topically, i.e. to the surface ofthe wound site, or subcutaneously. Using ECS cells, and in particular,AMP cells and preferably ACCS derived from AMP cells, all wound types,mechanical or thermal, acute or chronic, infected or sterile, orcongenital, undergo healing more rapidly than similar wounds left toheal naturally or which are treated with currently available methods.

The compositions and methods of the present invention are effective inaccelerating wound healing of wounds caused by a number of sources,including but not limited to incisional, compression, thermal, acute,chronic, infected, sterile and congenital injuries.

In addition to accelerating wound healing, the compositions of theinvention prevent and/or reduce the incidence of wound failure, such ashernia formation, by increasing both breaking strength and tensilestrength of wounds as well as increasing the rate in which increasedbreaking strength and tensile strength is attained during the woundhealing process. Thus, wounds not only heal faster, but become strongerfaster than wounds treated with other available agents or untreated.

Importantly, it has been discovered that the AMP cells and ACCS of theinvention are able to accelerate the rate of wound healing (includingincreased wound strength) in both non-contaminated and contaminated(infected) wounds. It is long known in the art that infected woundseither do not heal or the rate of healing is very slow. However, usingthe novel compositions and methods described herein, Applicants havefound that the rate of wound healing is accelerated even when the woundis infected. This unique ability to heal the wounds in the face ofinfection is not based on any antibacterial effect of the compositions,but rather is due to the unique combination of physiologically relevantcytokines secreted by the cells of the invention at physiologicallevels. The secretion of these physiologically relevant cytokines may beinto the extracellular space in which they are placed or into culturemedia to form ACCS. Such physiologically relevant cytokines includeVEGF, PDGF, Angiogenin, TGFβ2, TIMP-1 and TIMP-2. Because theeffectiveness of the AMP cell and in particular the ACCS compositionsare due to this unique cytokine profile, it is believed that any ECScomposition that produces a comparable cytokine profile will be equallyeffective and is thus contemplated by the instant invention.

These cytokines are known to be involved in many physiological processesincluding wound healing. VEGF and Angiogenin are both involved inregulating angiogenesis and vascularization. PDGF is involved inregulating cell growth and division and, like VEGF and Angiogenin, playsa significant role in angiogenesis. TGFβ2 is a member of the TGFsuperfamily, a group of cytokines that play a number of different rolesin many cellular functions. TIMP-1 and TIMP-2 are tissue inhibitors ofmetalloproteinases (MMPs). MMPs are a family of inflammatory cytokinesthat are present in high levels in non-healing wounds and are thought tointerfere with wound healing by destroying cytokines and other proteinsessential to the wound healing process. Previous studies havedemonstrated that the ratio of MMP-9 to TIMP-1 in wound fluids isinversely correlated with the healing of pressure wounds (Ladwig, GP, etal. Wound Rep Reg 2002, 10:26-37). Applicants have discovered that thephysiologically relevant levels of TIMP-1 and TIMP-2 secreted by thecells of the invention, in particular AMP cells, and found in, forexample, ACCS, block MMP activity and thus promote accelerated woundhealing.

The compositions of the invention are applied in a therapeuticallyeffective amount to accomplish accelerated wound healing, includingincreased wound strength and decreased wound failure. A “therapeuticallyeffective amount” of a therapeutic agent within the meaning of thepresent invention will be determined by a patient's attending physicianor veterinarian. Such amounts are readily ascertained by one of ordinaryskill in the art and will enable accelerated wound healing whenadministered in accordance with the present invention. Factors whichinfluence what a therapeutically effective amount will be include, thespecific activity of the therapeutic agent being used, the wound type(mechanical or thermal, full or partial thickness, etc.), the size ofthe wound, the wound's depth (if full thickness), the absence orpresence of infection, time elapsed since the injury's infliction, andthe age, physical condition, existence of other disease states, andnutritional status of the patient. Additionally, other medication thepatient may be receiving will effect the determination of thetherapeutically effective amount of the therapeutic agent to administer.

In addition, compositions of the invention may play a role in moresubstantial wound healing, such as in the regeneration of limbs.US2003/0212024, which is incorporated by reference herein, sets forthmethods of testing for such ability by measuring regeneration in thezebrafish, which is capable of complete regeneration followingamputation of the distal fin. Following amputation, completeregeneration occurs in several steps, including formation of a woundepidermis, migration of fibroblasts and scleroblasts (or osteoblasts)toward the wound epidermis, formation of a blastema, and outgrowth ofthe blastema via cell division and differentiation of the proximalportion of the fin to form specific structures of the regenerated fin.

In a preferred embodiment of the present invention, ECS cells andconditioned media derived therefrom, including AMP cells and/or ACCSderived therefrom, and/or cell lysates thereof should be topicallyadministered to the wound site to promote accelerated wound healing inthe patient. This topical administration can be as a single dose or asrepeated doses given at multiple designated intervals. It will readilybe appreciated by those skilled in the art that the preferred dosageregimen will vary with the type and severity of the injury beingtreated.

Formulations suitable for topical administration in accordance with thepresent invention comprise therapeutically effective amounts of thetherapeutic agent with one or more pharmaceutically acceptable carriersand/or adjuvants. ECS cells and conditioned media derived therefrom,including AMP cells and/or ACCS derived therefrom, and/or cell lysatesthereof may be used in conjunction with a variety of materials routinelyused in the treatment of wounds, such as collagen based creams, films,microcapsules, or powders; hyaluronic acid or otherglycosaminoglycan-derived preparations; creams, foams, suture material;and wound dressings. Alternatively, the ECS cells and conditioned mediaderived therefrom, including AMP cells and/or ACCS derived therefrom,and/or cell lysates thereof can be incorporated into a pharmaceuticallyacceptable solution designed for topical administration.

Reconstructive and Cosmetic Surgery

The compositions and methods of the present invention are effective inaccelerating healing following reconstructive and cosmetic surgery. It'sestimated that more that one million reconstructive procedures areperformed by surgeons every year. The goals of reconstructive surgerydiffer from those of cosmetic surgery. Reconstructive surgery isperformed on abnormal structures of the body, caused by birth defects,developmental abnormalities, trauma or injury, infection, tumors, ordisease. It is generally performed to improve function, but may also bedone to approximate a normal appearance. Cosmetic surgery is performedto reshape normal structures of the body to improve the patient'sappearance and self-esteem (i.e. rhinoplasty).

Reconstructive surgery is used to correct congenital deformities (i.e.birth defects such as birthmarks; cleft-lip and palate; syndactyly(webbed fingers); extra or absent fingers; and abnormal breastdevelopment) and deformities acquired as a result of accident,infection, disease, or in some cases, aging (i.e. burn wounds,lacerations, growths, and aging problems such as drooping eyelids whichcan impair vision). In each case, the reconstructive surgery requiresnot only repair of the surgical wounds, but often regeneration of tissue(i.e. bone or cartilage) and/or tissue growth to incorporate implantmaterial, etc.

Keloid and/or Hypertrophic Scarring

The compositions and methods of the present invention are also effectivein preventing or treating keloid and/or hypertrophic scars. Keloidand/or hypertrophic scars are abnormal scars that grow beyond theboundary of the original site of a skin injury. Although anyone can forma keloid and/or hypertrophic scar some ethnic groups are at more risk ofdeveloping them (i.e. keloid and/or hypertrophic scars are more commonin pigmented ethnic groups rather than in Caucasians). It is not fullyunderstood why or how keloid and/or hypertrophic scars occur Skin traumaappears to be the most common factor although they can form even whenthere appears to be no apparent cause Skin and/or muscle tension seem tocontribute to keloid and/or hypertrophic scar formation and this isdemonstrated by the most common sites of their formation (the upper armand back). However other factors are involved. Infection at a woundsite, repeated trauma to the same area or a foreign body in a wound canalso be factors. There appears to be a genetic component to keloidand/or hypertrophic scarring and individuals whose family members formkeloid and/or hypertrophic scars are at an increased risk of formingthem themselves. Other theories for the causes of keloid and/orhypertrophic scarring include a deficiency or an excess in melanocytehormone (MSH), decreased percentages of mature collagen and increasedsoluble collagen, or that very small blood vessels get blocked and theresulting lack of oxygen contribute to keloid and/or hypertrophic scarformation.

Current treatments for keloid and/or hypertrophic scars include surgicalremoval, non-surgical interventions and combination treatments. Surgicaltreatment of keloid and/or hypertrophic scars is the most effective andthe least complex of the available forms of treatment, although therecurrence rate is high. Lasers have been tried as an alternative totraditional surgery but so far the outcomes are no better. Non surgicaltreatments for keloid and/or hypertrophic scars include interferontherapy and have been reported as effective in reducing keloid and/orhypertrophic scarring. However, such treatment has significant sideeffects (i.e. toxicity, flu-like symptoms, depression, nausea andvomiting). Prolonged compression of scar tissue can theoretically softenand break up keloid and/or hypertrophic scars, however the practicalityof this option depends on the location of the keloid and/or hypertrophicscars. Other non-surgical interventions that are currently being triedwith varying results include antihistamines, vitamins, nitrogen mustard,Verapamil, retinoic acids. Combined treatments for keloid and/orhypertrophic scarring include surgical removal of scar tissue incombination with a steroid injection. This type of treatment isvariously reported as having between a 50% to 70% rate of recurrence.Another option combines surgery with external type radiotherapy.Radiation has the effect of interfering with skin growth (fibroblasts)and collagen production. Research varies on which type of combinationtherapy is the more effective.

Surgical Adhesions.

The compositions and methods of the present invention are also effectivein preventing or treating surgical adhesions. Adhesions are internalscars made of strand like fibrous tissue that forms an abnormal bondbetween two parts of the body after trauma, injury, and/or surgery andin some cases may cause severe clinical consequences including severepelvic pain, infertility and intestinal obstruction.

Any peritoneal injury can result in fibrous adhesion formation. Forexample, infection, endometriosis, chemotherapy, radiation and cancermay damage tissue and initiate adhesion formation. However, the mostcommon cause of adhesion formation is surgery. Adhesions normally occurat the site of the surgical procedure and are a result of the body'snormal healing process. Surgical procedures most commonly associatedwith adhesion formation are ovarian cystectomy, myomectomy, totalabdominal hysterectomy, salpingostomy/fimbrioplasty, excision ofendometriosis, excision of eptopic pregnancy, cesarean section, andadhesiolysis.

Following reproductive pelvic surgery performed by laparotomy, more than50% of patients are shown to have adhesions at subsequent surgeries. Thenumber of hospital readmissions for adhesion-related complicationsrivals the number of operations for heart bypass, hip replacements andappendectomies. It is not unusual for several organs to be adhered toeach other causing traction (pulling) of nerves. Nerve endings may alsobecome entrapped within a developing adhesion causing severe pain.Intestinal obstruction is one of the most severe consequences ofadhesions. Adhesions can form elsewhere such as around the heart, spineand in the hand where they may lead to other problems.

Dental Applications.

The compositions and methods of the present invention are also effectivein the treatment of many dental diseases and disorders including, forexample, periodontal disease and healing following tooth extraction.Periodontal disease is common in the United States and throughout theworld, and is a significant public health issue in many areas. Damagethat occurs early in the course of disease, such as that caused bygingivitis or moderate periodontitis, can often be reversed or at leastits progression stopped by aggressive treatment, including rootscraping, surgery and antibiotic treatments. However, with more advancedperiodontal disease it is usually impossible to effectively treat and,therefore, requires the rebuilding of portions of the tooth and rootsystem and the upper and lower jaw bones with synthetic materials, or bybone or skin grafts. While these treatments have improved significantly,but they do not bring teeth, bone and gums back to normal. As aconsequence, patients continue to have difficulties eating, may havedistorted jaws. It is also possible the dental disease can lead to othermedical problems, including infections and heart problems.

Peptic Ulcer Disease (PUD)

The compositions and methods of the present invention are also effectivein the treatment of peptic ulcer disease. A peptic ulcer is an ulcer ofone of those areas of the gastrointestinal tract that are usuallyacidic. Most peptic ulcers arise in the duodenum rather than in thestomach. Furthermore, most peptic ulcers are associated withHelicobacter pylori, a spiral-shaped bacterium that lives in the acidicenvironment of the stomach. Ulcers can also be caused or worsened bydrugs such as Aspirin and other non-steroidal anti-inflammatory drugs.

Dermatological Applications and Cosmeceuticals.

The same properties that make ECS cells and conditioned media derivedtherefrom, including AMP cells and/or ACCS derived therefrom, and/orcell lysates thereof, alone or in combination, as well as compositionsof ECS cells as defined herein useful for wound healing make themsimilarly well-suited for the treatment of cosmetic and/ordermatological conditions, including aging skin. The dermal layer ofskin, important in maintaining the elasticity and appearance of theskin, thins with age, leading to sagging and wrinkles.

As described above, fetal skin has much more effective repair mechanismsthan adult skin, and, once wounded, in the first two trimesters ofpregnancy, it is able to heal without the formation of scars. Thiscapability does appear to require the fetal immune system, fetal serum,or amniotic fluid (Bleacher J C, et al., J Pediatr Surg 28: 1312-4,1993); Ihara S, Motobayashi Y., Development 114: 573-82. 1992). Suchabilities of fetal tissue have led to the suggested use of compoundsproduced by fetal tissue for regenerating and/or improving theappearance of skin (see, for example, US 2004/0170615, which isincorporated by reference in its entirety herein).

The present invention contemplates the use of the ECS cells andconditioned media derived therefrom, including AMP cells and/or ACCSderived therefrom, and/or cell lysates thereof, in the use of novelcosmetic skin care compositions. Such compounds may be delivered to skinby way of, but not limited to, a solution, a lotion, an ointment, acream, a gel, or a skin peelable strip.

The methods generally include the step of topically applying a safe andeffective amount of the composition to the skin of a mammal in needthereof. Additional skin care components, as well as cosmeticallyacceptable, dermatologically acceptable or pharmaceutically acceptablecarriers may be included in such compositions.

Cosmetic compositions usually comprise an aqueous phase that is gelled,i.e. thickened, using one or more thickener(s) or gelling agent(s).These may be, for example, lotions which are aqueous solutions notcontaining an oily phase, or emulsions which may be direct oil-in-wateremulsions including a fatty phase or oily phase dispersed in an aqueouscontinuous phase, or water-in-oil reverse emulsions including an aqueousphase dispersed in an oily continuous phase. The term “emulsions” meansherein both the dispersions obtained in the absence of emulsifyingsurfactants and the emulsions obtained in the presence of emulsifyingsurfactants.

Oil-in-water emulsions are the emulsions most frequently sought incosmetics due to the fact that, when applied to the skin, they give asofter, less greasy, fresher and lighter feel than water-in-oil emulsionsystems, by virtue of the presence of water in the continuous outerphase.

The nature of the compounds used for gelling the aqueous phase and theircontent in the composition are chosen as a function of the desired typeof texture, which may range from fluid lotions to more or less thickemulsions that may constitute milks or creams. The main thickeners orgelling agents used in cosmetics are chosen from the following compoundsnatural polymers such as xanthan gum and guar gum or cellulosederivatives, starches and alginates and crosslinked polymeric gellingagents such as the Carbopols or crosslinked and at least partiallyneutralized 2-acrylamido-2-methylpropanesulfonic acid polymers.

Differentiation of ECS Cells, Including AMP Cells, and DifferentiatedCell Types

The ECS cells, including AMP cells, may be contacted with various growthfactors (termed differentiation factors) that influence differentiationof such stem cells into particular cell types such as skin cells, musclecells, bone cells and nerve cells.

The literature is replete with differentiation protocols for embryonicas well as non-embryonic stem or other multipotent cells, including stemcells (see for example osteogenic differentiation (Shi, Y. Y., et al.,(2005) Plast Reconstr Surg 116, 1686-96); adipogenic differentiation(Shi, Y. Y., et al., (2005) Plast Reconstr Surg 116, 1686-96);chondrogenic differentiation (Malladi, P., et al., (2006) Am J PhysiolCell Physiol 290, C1139-46). One skilled in the art will recognize thatany of these protocols may be applied to the ECS cell compositions,including the AMP cell compositions described herein to producepartially or fully differentiated cells for such uses.

Differentiated cells derived from ECS cells including AMP cells may bedetected and/or enriched by the detection of tissue-specific markers byimmunological techniques, such as flow immunocytochemistry forcell-surface markers, immunohistochemistry (for example, of fixed cellsor tissue sections) for intracellular or cell-surface markers, Westernblot analysis of cellular extracts, and enzyme-linked immunoassay, forcellular extracts or products secreted into the medium. The expressionof tissue-specific gene products can also be detected at the mRNA levelby Northern blot analysis, dot-blot hybridization analysis, or byreverse transcriptase initiated polymerase chain reaction (RT-PCR) usingsequence-specific primers in standard amplification methods.

Alternatively, differentiated cells may be detected using selectionmarkers. For example, AMP cells can be stably transfected with a markerthat is under the control of a tissue-specific regulatory region as anexample, such that during differentiation, the marker is selectivelyexpressed in the specific cells, thereby allowing selection of thespecific cells relative to the cells that do not express the marker. Themarker can be, e.g., a cell surface protein or other detectable marker,or a marker that can make cells resistant to conditions in which theydie in the absence of the marker, such as an antibiotic resistance gene(see e.g., in U.S. Pat. No. 6,015,671).

Isolation, Identification and Characterization of ECS Cells IncludingAMP Cells

Various methods for isolating cells from the extraembryonic tissue,which may then be used to produce the ECS cells of the instant inventionare described in the art (see, for example, US2003/0235563,US2004/0161419, US2005/0124003, U.S. Provisional Application Nos.60/666,949, 60/699,257, 60/742,067, 60/813,759, U.S. application Ser.No. 11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pat. No. 7,045,148, US2004/0048372,and US2003/0032179).

Identifying ECS cells—Once extraembryonic tissue is isolated, it isnecessary to identify which cells in the tissue have the characteristicsassociated with ECS cells (see definition above). For example, cells areassayed for their ability to secrete a unique combination of cytokinesinto the extracellular space or into surrounding culture media. Suitablecells are those in which the cytokine or cytokines occurs in thephysiological range of ˜5.0-16 ng/ml for VEGF, ˜3.5-4.5 ng/ml forAngiogenin, ˜100-165 pg/ml for PDGF, ˜2.5-2.7 ng/ml for TGFβ2, ˜0.68 μgml for TIMP-1 and ˜1.04 μg/ml for TIMP-2.

In a particular embodiment, AMP cell compositions are prepared using thesteps of a) recovery of the amnion from the placenta, b) dissociation ofthe cells from the amniotic membrane, c) culturing of the cells in abasal medium with the addition of a naturally derived or recombinantlyproduced human protein; and optionally d) further proliferation of thecells using additional additives and/or growth factors. Details arecontained in US Publication No. 2006-0222634-A1, which is incorporatedherein by reference.

In a preferred embodiment, the following method is used to obtainselected AMP cells. The cells are plated into plastic tissue culturevessels (i.e. T75 flasks) immediately upon isolation from the amnion.After ˜1-5 days, preferably ˜1-3 days, and most preferably ˜2 days inculture, non-adherent cells are removed from the plastic tissue culturevessel and discarded and the adherent cells are kept. This attachment ofcells to a plastic tissue culture vessel is the selection method used toobtain the desired population of AMP cells. Adherent and non-adherentAMP cells appear to have similar cell surface marker expression profilesbut the adherent cells have the advantage of possessing greaterviability than the non-adherent population of cells and are thus thedesired population of AMP cells. Adherent AMP cells are cultured untilthey reach ˜13,000-700,000 cells/cm², preferably ˜53,000-500,000cells/cm² and most preferably ˜120,000-300,000 cells/cm². At this point,the cultures are confluent or close to confluent. Suitable cellscultures will reach this number of cells between ˜5-14 days, preferablybetween 5-9 days. Attaining this criterion is an indicator of theproliferative potential of the AMP cells and cells that do not achievethis criterion are not selected for further analysis and use. Once theAMP cells reach ˜13,000-700,000 cells/cm², preferably ˜53,000-500,000cells/cm² and most preferably ˜120,000-300,000 cells/cm², they areremoved from the plastic tissue culture vessel and cryopreserved. Thiscollection time point is called p0 and all subsequent analyses are donewith thawed p0 AMP cells.

The AMP cells of the invention are characterized by assaying forphysiologically relevant cytokines Suitable cells are those in whicheach cytokine occurs in the physiological range of ˜5.0-16 ng/ml forVEGF, ˜3.5-4.5 ng/ml for Angiogenin, ˜100-165 pg/ml for PDGF, ˜2.5-2.7ng/ml for TGFβ2, ˜0.68 μg/ml for TIMP-1 and ˜1.04 μg/ml for TIMP-2.

In addition, the AMP cells of the invention are further characterized asfollows: Using commercially available antibodies to known stem cellmarkers, freshly isolated AMP cells have been extensively characterized.Briefly, freshly isolated AMP cells are substantially negative withrespect to CD90 and CD117. In addition, such cell populations areessentially negative for protein expression of CD34, CD44, CD45, CD140b,CD105; essentially positive for protein expression of CD9 and CD29;between about 70-95% positive for protein expression of SSEA4, CD10,CD166 and CD227; between about 60-95% positive for protein expression ofHLA-G, EGFR and CD26; and between about 10-50% positive for proteinexpression of CD71. Details on this procedure are contained in USPublication No. 2006-0222634-A1, which is incorporated herein byreference.

In alternative embodiments substantially purified AMP cell populationscan be created using antibodies against protein markers expressed(positive selection) or not expressed (negative selection) on the cellsurface of the AMP cells. These antibodies may be used to identify,characterize, isolate or create such substantially purified populationsof AMP cells expressing those protein markers using a variety ofmethods. Details on this procedure are contained in US Publication No.2006-0222634-A1, which is incorporated herein by reference.

In addition, protein markers that are not expressed on the surface ofAMP cells may also be used to identify, isolate or create populations ofAMP cells not expressing those markers. Such procedures may involve anegative selection method, such as passage of sample cells over a columncontaining anti-protein marker antibodies or by binding of cells tomagnetic bead-conjugated antibodies to the protein markers or by panningon plates coated with protein marker antibodies and collecting theunbound cells. Alternatively, a single-cell suspension may be exposed toone or more fluorescent-labeled antibodies that immuno-specifically bindto the protein markers. Details on this procedure are contained in USPublication No. 2006-0222634-A1, which is incorporated herein byreference.

Expanded Populations of ECS Cells, Including AMP Cells.

One of skill in the art will recognize that any of the ECS cells of theinstant invention may be expanded using the methods described below.

As described herein and in US Publication No. 2006-0222634-A1, which isincorporated herein by reference, Applicants have discovered a novelmethod for isolation and propagation of multipotent, AMP cells. Suchmethods result in AMP cell compositions which are expanded formultipotent cells, thereby providing, for the first time, sufficientquantities of cells to enable therapeutic cell transplantation. ExpandedAMP cell compositions, which are made in accordance with the subjectinvention, are compositions in which the level of cells per gram ofamniotic tissue is at least 50 fold and up to 150 fold higher after 5passages, as compared to about 20 fold using previous methods.Alternatively, expanded AMP cell compositions, which are made inaccordance with the subject invention, are compositions in which thelevel of cells per gram of amniotic tissue is at least 30 fold and up to100 fold higher after 3 passages.

Additionally, the methods used for cell culture and proliferationprovide a means to culture the cells, as well as other cells includingbut not limited to multipotent, pluripotent cells or totipotent cells,including, but not limited to, embryonic stem cells, in an animal-freesystem. Furthermore, the culture conditions described provide a cellthat is less dependent on attachment to a culture surface for viability,thus allowing for propagation of the cells using suspension culture forefficient scale-up. Details on this procedure are contained in USPublication No. 2006-0222634-A1, which is incorporated herein byreference.

The expanded AMP cell compositions described herein demonstrateextensive proliferative potential, express certain genes known to beexpressed only in undifferentiated cells (i.e. Nanog and Oct-4) and candifferentiate into cell types that normally arise from all threeembryonic germ layers (endoderm, ectoderm and mesoderm). Thisdifferentiation potential suggests that these expanded AMP cells may beable to contribute to a variety of cell types. The AMP cell compositionsdescribed herein are also useful as feeder layers for the growth of avariety of cell types, including but not limited to embryonic stem cells(ES cells). AMP cells, including those described herein, also produce awide variety of cytokines and growth factors, thereby making both thecell compositions, conditioned media derived from the cells (ACCS), celllysates therefrom, extracellular matrices produced by the cells, andcombinations thereof useful for a variety of therapeutic applications,in particular cell-based therapeutic applications such astransplantation therapies.

Culturing of the AMP cells

The cells are cultured in a basal medium. Such medium includes, but isnot limited to, Epilife (Cascade Biologicals), Opti-pro, VP-SFM, IMDM,Advanced DMEM, K/O DMEM, 293 SFM II (all made by Gibco; Invitrogen),HPGM, Pro 293S-CDM, Pro 293A-CDM, UltraMDCK, UltraCulture (all made byCambrex), Stemline I and Stemline II (both made by Sigma-Aldrich), DMEM,DMEM/F-12, Ham's F12, M199, and other comparable basal media. Such mediashould either contain human protein or be supplemented with humanprotein. As used herein a “human protein” is one that is producednaturally or one that is produced using recombinant technology. “Humanprotein” also is meant to include a human fluid or derivative orpreparation thereof, such as human serum or amniotic fluid, whichcontains human protein. Details on this procedure are contained in USPublication No. 2006-0222634-A1, which is incorporated herein byreference.

In a most preferred embodiment, the cells are cultured using a systemthat is free of animal products to avoid xeno-contamination. In thisembodiment, the culture medium is Stemline I or II, Opti-pro, or DMEM,with human albumin added up to concentrations of 10%. Alternatively,UltraCulture may be used, with substitution of transferrin with humanrecombinant transferrin, and replacement of the bovine albumin (BSA)with human albumin at concentrations of up to 10%. The invention furthercontemplates the use of any of the above basal media whereinanimal-derived proteins are replaced with recombinant human proteins andanimal-derived serum, such as BSA, is replaced with human albumin. Inpreferred embodiments, the media is serum-free in addition to beinganimal-free. Details on this procedure are contained in US PublicationNo. 2006-0222634-A1, which is incorporated herein by reference.

In alternative embodiments, where the use of non-human serum is notprecluded, such as for in vitro uses, the culture medium may besupplemented with serum derived from mammals other than humans, inranges of up to 40%.

Additional Proliferation

Optionally, other proliferation factors are used. In one embodiment,epidermal growth factor (EGF), at a concentration of between 0-1 μg/mlis used. In a preferred embodiment, the EGF concentration is around 10ng/ml. Alternative growth factors which may be used include, but are notlimited to, TGFα or TGFβ (5 ng/ml; range 0.1-100 ng/ml), activin A,cholera toxin (preferably at a level of about 0.1 μg/ml; range 0-10μg/ml), transferrin (5 μg/ml; range 0.1-100 μg/ml), fibroblast growthfactors (bFGF 40 ng/ml (range 0-200 ng/ml), aFGF, FGF-4, FGF-8; (all inrange 0-200 ng/ml), bone morphogenic proteins (i.e. BMP-4) or othergrowth factors known to enhance cell proliferation.

Generation of ACCS—The AMP cells of the invention can be used togenerate ACCS. In one embodiment, the AMP cells are isolated asdescribed herein and 10×10⁶ cells are seeded into T75 flasks containingbetween 5-30 ml culture medium, preferably between 10-25 ml culturemedium, and most preferably about 10 ml culture medium. The cells arecultured until confluent, the medium is changed and in one embodimentthe ACCS is collected 1 day post-confluence. In another embodiment themedium is changed and ACCS is collected 2 days post-confluence. Inanother embodiment the medium is changed and ACCS is collected 4 dayspost-confluence. In another embodiment the medium is changed and ACCS iscollected 5 days post-confluence. In a preferred embodiment the mediumis changed and ACCS is collected 3 days post-confluence. Skilledartisans will recognize that other embodiments for collecting ACCS fromconfluent cultures, such as using other tissue culture vessels,including but not limited to cell factories, flasks, hollow fibers, orsuspension culture apparatus, are also contemplated by the methods ofthe invention. It is also contemplated by the instant invention that theACCS be cryopreserved following collection.

Compositions

The compositions of the invention include substantially purifiedpopulations of ECS cells, conditioned media derived therefrom, celllysates derived therefrom and cell products derived therefrom, andpharmaceutical compositions of such. In preferred embodiments, thesubstantially purified populations of ECS cells are AMP cells, ACCS,cell lysates derived therefrom, cell products derived therefrom, andpharmaceutical compositions of such. The compositions of the inventioncan be prepared in a variety of ways depending on the intended use ofthe compositions. For example, a composition useful in practicing theinvention may be a liquid comprising an agent of the invention, i.e. asubstantially purified population of AMP cells, in solution, insuspension, or both (solution/suspension). The term“solution/suspension” refers to a liquid composition where a firstportion of the active agent is present in solution and a second portionof the active agent is present in particulate form, in suspension in aliquid matrix. A liquid composition also includes a gel. The liquidcomposition may be aqueous or in the form of an ointment, salve, cream,or the like. In a particularly preferred embodiment, the liquidcomposition may contain ACCS.

An aqueous suspension or solution/suspension useful for practicing themethods of the invention may contain one or more polymers as suspendingagents. Useful polymers include water-soluble polymers such ascellulosic polymers and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. An aqueous suspension orsolution/suspension of the present invention is preferably viscous ormuco-adhesive, or even more preferably, both viscous and muco-adhesive.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions ofsubstantially purified populations of ECS cells, conditioned mediaderived therefrom, cell lysates derived therefrom and cell productsderived therefrom, and a pharmaceutically acceptable carrier. Thepresent invention also provides pharmaceutical compositions ofsubstantially purified populations of AMP cells, ACCS, cell lysatesderived therefrom, cell products derived therefrom, and apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the composition is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin, and still others are familiar to skilledartisans.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Treatment Kits

The invention also provides for an article of manufacture comprisingpackaging material and a pharmaceutical composition of the inventioncontained within the packaging material, wherein the pharmaceuticalcomposition comprises a substantially purified population of ECS cells,conditioned media derived therefrom, cell lysates derived therefrom andcell products derived therefrom. In preferred embodiments, thesubstantially purified populations of cells are AMP cells, ACCS, celllysates derived therefrom, cell products derived therefrom. In aparticularly preferred embodiment, the composition is ACCS. Thepackaging material comprises a label or package insert which indicatesthat the substantially purified population of ECS cells, conditionedmedia derived therefrom, cell lysates derived therefrom and cellproducts derived therefrom, or AMP cells, ACCS derived therefrom, celllysates derived therefrom and cell products derived therefrom can beused for treating a variety of disorders including but not limited toaccelerating wound healing, preventing or reducing wound healingfailure, scarring, etc.

Formulation, Dosage and Administration

Compositions comprising ECS cells, conditioned media derived therefrom,cell lysates derived therefrom and cell products derived therefrom, andin preferred embodiments substantially purified populations of cells areAMP cells, ACCS, cell lysates derived therefrom, cell products derivedtherefrom, may be administered to a subject to provide various cellularor tissue functions, for example, to accelerate wound healing. As usedherein “subject” may mean either a human or non-human animal.

Such compositions may be formulated in any conventional manner using oneor more physiologically acceptable carriers optionally comprisingexcipients and auxiliaries. Proper formulation is dependent upon theroute of administration chosen. The compositions may be packaged withwritten instructions for their use in wound healing, tissueregeneration, or restoring a therapeutically important metabolicfunction. The compositions may also be administered to the recipient inone or more physiologically acceptable carriers. Carriers for the cellsmay include but are not limited to solutions of phosphate bufferedsaline (PBS) or lactated Ringer's solution containing a mixture of saltsin physiologic concentrations.

One of skill in the art may readily determine the appropriateconcentration, or dose, of ECS cells, including AMP cells and/or ACCSand/or lysates and/or cell products derived therefrom, for a particularpurpose. The skilled artisan will recognize that a preferred dose is onewhich produces a therapeutic effect, such as accelerating wound healing,in a patient in need thereof. Determination of a preferred dose is basedon the specific activity of the ECS cells, including AMP cells, andACCS. Specific activity can be readily determined by assaying thepotential of AMP cells and/or ACCS to stimulate cell proliferation instandard proliferation assays which are well known to skilled artisans(see, for example, Nissen, N. N., et al., J Trauma 2003; 54:1205-1211,incorporated herein in its entirety). Further, proper doses of ECScells, including AMP cells and/or ACCS and/or lysates and/or cellproducts derived therefrom will require empirical determination at timeof use based on several variables including but not limited to theseverity and type of wound being treated; patient age, weight, sex,health; other medications and treatments being administered to thepatient; and the like. For AMP cells a preferred dose is in the range ofabout 10-300,000 cells/μl vehicle. Another preferred dose is in therange of about 100-30,000 cells/μl vehicle. Another preferred dose is inthe range of about 1000-3000 cells/μl vehicle. In a particular preferredembodiment, it has been found that relatively small amounts of AMP cellscan accelerate wound healing, etc. One of skill in the art will alsorecognize that number of doses (dosing regimen) to be administered needsalso to be empirically determined based on, for example, severity andtype of wound being treated. In a preferred embodiment, one dose issufficient to accelerated wound healing. Other preferred embodimentscontemplate, 2, 3, 4, or more doses to accelerate wound healing, etc.

In addition, one of skill in the art may readily determine theappropriate concentration, or dose, of conditioned media, including, forexample, ACCS, for a particular purpose. A preferred dose is in therange of about 0.5-2000 μl/cm². Another preferred dose is in the rangeof about 5-1000 μl/cm². Another preferred dose is in the range of about50-100 μl/cm². In a particularly preferred embodiment, it has been foundthat relatively small amounts of ACCS can accelerate wound healing, etc.One of skill in the art will also recognize that number of doses (dosingregimen) to be administered needs also to be empirically determinedbased on, for example, severity and type of wound being treated. In apreferred embodiment, one dose is sufficient to accelerated woundhealing. Other preferred embodiments contemplate, 2, 3, 4, or more dosesto accelerate wound healing, etc.

Skilled artisans will recognize that any and all of the standard methodsand modalities for wound healing currently in clinical practice andclinical development are suitable for practicing the methods of theinvention. Routes of administration, formulation, co-administration withother agents (if appropriate) and the like are discussed in detailelsewhere herein.

Timing of treatment and administration of the compositions of theinvention is also dependent upon the severity and type of wound beingtreated. For example, for surgical wounds it may be advantageous totreat, or prime, the surgical site prior to incisional injury. It mayalso be advantageous to treat the surgical site post-surgery. It mayalso be advantageous to treat the surgical site during surgery. Otherembodiments contemplate different dosing intervals (i.e. prior tosurgery and/or during surgery and/or after surgery). In the case oftraumatic wounds, when treatment is only possible after the injury hasoccurred, it may be advantageous to treat the wound immediately uponpresentation and again following medical and/or surgical intervention.Attending physicians will determine the exact treatment regimen based onthe severity of the wound being treated, etc.

It may be desirable to administer ECS cells and/or conditioned mediaderived therefrom and/or cell lysates derived therefrom and/or cellproducts derived therefrom, including AMP cells, and/or ACCS and/or celllysates derived therefrom and/or cell products derived therefrom incombination with other agents, including active agents and/or inactiveagents. Active agents include but are not limited to growth factors,cytokines, chemokines, antibodies, antibiotics, anti-fungals,anti-virals, other cell types, and the like. Inactive agents includecarriers, diluents, stabilizers, gelling agents, delivery vehicles, ECMs(natural and synthetic), scaffolds, and the like.

ECS cells and/or conditioned media derived therefrom and/or cell lysatesderived therefrom and/or cell products derived therefrom, including AMPcells, and/or ACCS and/or cell lysates derived therefrom and/or cellproducts derived therefrom can be administered by injection into atarget site of a subject, preferably via a delivery device, such as atube, e.g., catheter. In a preferred embodiment, the tube additionallycontains a needle, e.g., a syringe, through which the cells can beintroduced into the subject at a desired location. Specific,non-limiting examples of administering cells to subjects may alsoinclude administration by subcutaneous injection, intramuscularinjection, or intravenous injection. For example, if administration isintravenous, an injectable liquid suspension of AMP cells and/or ACCSand/or lysates and/or cell products derived therefrom can be preparedand administered by a continuous drip or as a bolus.

ECS cells and/or conditioned media derived therefrom and/or cell lysatesderived therefrom and/or cell products derived therefrom, including AMPcells, and/or ACCS and/or cell lysates derived therefrom and/or cellproducts derived therefrom may also be inserted into a delivery device,e.g., a syringe, in different forms. For example, the cells can besuspended in a solution contained in such a delivery device. As usedherein, the term “solution” includes a pharmaceutically acceptablecarrier or diluent in which the cells of the invention remain viable.Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. The solution ispreferably sterile and fluid to the extent that easy syringabilityexists. Preferably, the solution is stable under the conditions ofmanufacture and storage and preserved against the contaminating actionof microorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. Solutions of the invention can be prepared by incorporatingcompositions as described herein, in a pharmaceutically acceptablecarrier or diluent and, as required, other ingredients enumerated above,followed by filter sterilization.

Alternatively, ECS cells including AMP cells may be transplanted intothe recipient where the cells will proliferate and differentiate to formnew cells and tissues thereby providing the physiological processesnormally provided by that tissue, or may produce factors that cause themigration and/or differentiation of cells in the area of the transplant.Tissues are an aggregation of similarly specialized cells united in theperformance of a particular function. Tissue is intended to encompassall types of biological tissue including both hard and soft tissue. Softtissue refers to tissues that connect, support, or surround otherstructures and organs of the body. Soft tissue includes muscles, tendons(bands of fiber that connect muscles to bones), fibrous tissues, fat,blood vessels, nerves, and synovial tissues (tissues around joints).Hard tissue includes connective tissue (e.g., hard forms such as osseoustissue or bone) as well as other muscular or skeletal tissue.

Support matrices into which the ECS cells and/or conditioned mediaderived therefrom and/or cell lysates derived therefrom and/or cellproducts derived therefrom, including AMP cells, and/or ACCS and/or celllysates derived therefrom and/or cell products derived therefrom can beincorporated or embedded include matrices which are recipient-compatibleand which degrade into products which are not harmful to the recipient.These matrices provide support and protection for ECS cells includingAMP cells in vivo and are, therefore, the preferred form in which suchcells are transplanted into the recipient subjects.

Natural and/or synthetic biodegradable matrices are examples of suchmatrices. Natural biodegradable matrices include plasma clots, e.g.,derived from a mammal, collagen, fibronectin, and laminin matrices.Suitable synthetic material for a cell transplantation matrix must bebiocompatible to preclude migration and immunological complications, andshould be able to support extensive cell growth and differentiated cellfunction. It must also be resorbable, allowing for a completely naturaltissue replacement. The matrix should be configurable into a variety ofshapes and should have sufficient strength to prevent collapse uponimplantation. Recent studies indicate that the biodegradable polyesterpolymers made of polyglycolic acid fulfill all of these criteria(Vacanti, et al. J. Ped. Surg. 23:3-9 (1988); Cima, et al. Biotechnol.Bioeng. 38:145 (1991); Vacanti, et al. Plast. Reconstr. Surg. 88:753-9(1991)). Other synthetic biodegradable support matrices includesynthetic polymers such as polyanhydrides, polyorthoesters, andpolylactic acid. Further examples of synthetic polymers and methods ofincorporating or embedding cells into these matrices are also known inthe art. See e.g., U.S. Pat. Nos. 4,298,002 and 5,308,701.

Attachment of the cells to the polymer may be enhanced by coating thepolymers with compounds such as basement membrane components, agar,agarose, gelatin, gum arabic, collagens types I, II, III, IV and V,fibronectin, laminin, glycosaminoglycans, mixtures thereof, and othermaterials known to those skilled in the art of cell culture. Allpolymers for use in the matrix must meet the mechanical and biochemicalparameters necessary to provide adequate support for the cells withsubsequent growth and proliferation. The polymers can be characterizedwith respect to mechanical properties such as tensile strength using anInstron tester, for polymer molecular weight by gel permeationchromatography (GPC), glass transition temperature by differentialscanning calorimetry (DSC) and bond structure by infrared (IR)spectroscopy, with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies.

One of the advantages of a biodegradable polymeric matrix is thatbioactive compounds can be incorporated directly into the support matrixso that they are slowly released as the support matrix degrades in vivo.As the cell-polymer structure is vascularized and the structuredegrades, AMP cells may differentiate according to their inherentcharacteristics. Factors, including nutrients, growth factors, inducersof differentiation or de-differentiation (i.e., causing differentiatedcells to lose characteristics of differentiation and acquirecharacteristics such as proliferation and more general function),products of secretion, immuno-modulators, inhibitors of inflammation,regression factors, biologically active compounds which enhance or allowingrowth of the lymphatic network or nerve fibers, hyaluronic acid, anddrugs, which are known to those skilled in the art and commerciallyavailable with instructions as to what constitutes an effective amount,from suppliers such as Collaborative Research, Sigma Chemical Co.,vascular growth factors such as vascular endothelial growth factor(VEGF), epidermal growth factor (EGF), and heparin binding epidermalgrowth factor like growth factor (HB-EGF), could be incorporated intothe matrix or be provided in conjunction with the matrix. Similarly,polymers containing peptides such as the attachment peptide RGD(Arg-Gly-Asp) can be synthesized for use in forming matrices (see e.g.U.S. Pat. Nos. 4,988,621, 4,792,525, 5,965,997, 4,879,237 and4,789,734).

In another example, the undifferentiated, partially differentiated orfully differentiated ECS cells including AMP cells, may be transplantedin a gel matrix (such as Gelfoam from Upjohn Company) which polymerizesto form a substrate in which the cells can grow. A variety ofencapsulation technologies have been developed (e.g. Lacy et al.,Science 254:1782-84 (1991); Sullivan et al., Science 252:718-712 (1991);WO 91/10470; WO 91/10425; U.S. Pat. No. 5,837,234; U.S. Pat. No.5,011,472; U.S. Pat. No. 4,892,538). During open surgical procedures,involving direct physical access to the damaged tissue and/or organ, allof the described forms of undifferentiated, partially differentiated orfully differentiated ECS cell including AMP cell delivery preparationsare available options. These cells can be repeatedly transplanted atintervals until a desired therapeutic effect is achieved.

The present invention also relates to the use of ECS cells including AMPcells in three dimensional cell and tissue culture systems to formstructures analogous to tissue counterparts in vivo. The resultingtissue will survive for prolonged periods of time, and performtissue-specific functions following transplantation into the recipienthost. Methods for producing such structures are described in U.S. Pat.Nos. 5,624,840 and 6,428,802, which are incorporated herein in theirentireties.

The three-dimensional matrices to be used are structural matrices thatprovide a scaffold for the cells, to guide the process of tissueformation. Scaffolds can take forms ranging from fibers, gels, fabrics,sponge-like sheets, and complex 3-D structures with pores and channelsfabricated using complex Solid Free Form Fabrication (SFFF) approaches.Cells cultured on a three-dimensional matrix will grow in multiplelayers to develop organotypic structures occurring in three dimensionssuch as ducts, plates, and spaces between plates that resemblesinusoidal areas, thereby forming new tissue. Thus, in preferredaspects, the present invention provides a scaffold, multi-layer cell andtissue culture system. As used herein, the term “scaffold” means athree-dimensional (3D) structure (substrate and/or matrix) that cellsgrow in or on. It may be composed of biological components, syntheticcomponents or a combination of both. Further, it may be naturallyconstructed by cells or artificially constructed. In addition, thescaffold may contain components that have biological activity underappropriate conditions. The structure of the scaffold can include amesh, a sponge or can be formed from a hydrogel.

Examples of such scaffolds include a three-dimensional stromal tissue orliving stromal matrix which has been inoculated with stromal cells thatare grown on a three dimensional support. The extracellular matrixproteins elaborated by the stromal cells are deposited onto thescaffold, thus forming a living stromal tissue. The living stromaltissue can support the growth of ECS cells including AMP cells ordifferentiated cells later inoculated to form the three-dimensional cellculture. Examples of other three dimensional scaffolds are described inU.S. Pat. No. 6,372,494.

The design and construction of the scaffolding to form athree-dimensional matrix is of primary importance. The matrix should bea pliable, non-toxic, injectable porous template for vascular ingrowth.The pores should allow vascular ingrowth. These are generallyinterconnected pores in the range of between approximately 100 and 300microns, i.e., having an interstitial spacing between 100 and 300microns, although larger openings can be used. The matrix should beshaped to maximize surface area, to allow adequate diffusion ofnutrients, gases and growth factors to the cells on the interior of thematrix and to allow the ingrowth of new blood vessels and connectivetissue. At the present time, a porous structure that is relativelyresistant to compression is preferred, although it has been demonstratedthat even if one or two of the typically six sides of the matrix arecompressed, that the matrix is still effective to yield tissue growth.

The polymeric matrix may be made flexible or rigid, depending on thedesired final form, structure and function. For repair of a defect, forexample, a flexible fibrous mat is cut to approximate the entire defectthen fitted to the surgically prepared defect as necessary duringimplantation. An advantage of using the fibrous matrices is the ease inreshaping and rearranging the structures at the time of implantation.

A sponge-like structure can also be used to create a three-dimensionalframework. The structure should be an open cell sponge, one containingvoids interconnected with the surface of the structure, to allowadequate surfaces of attachment for sufficient ECS cells including AMPcells or differentiated cells to form a viable, functional implant.

The invention also provides for the delivery of ECS cells including AMPcells, including AMP cell compositions described herein, in conjunctionwith any of the above support matrices as well as amnion-derivedmembranes. Such membranes may be obtained as a by-product of the processdescribed herein for the recovery of AMP cells, or by other methods,such as are described, for example, in U.S. Pat. No. 6,326,019 whichdescribes a method for making, storing and using a surgical graft fromhuman amniotic membrane, US 2003/0235580 which describes reconstitutedand recombinant amniotic membranes for sustained delivery of therapeuticmolecules, proteins or metabolites, to a site in a host, U.S.2004/0181240, which describes an amniotic membrane covering for a tissuesurface which may prevent adhesions, exclude bacteria or inhibitbacterial activity, or to promote healing or growth of tissue, and U.S.Pat. No. 4,361,552, which pertains to the preparation of cross-linkedamnion membranes and their use in methods for treating burns and wounds.In accordance with the present invention, ECS cells including AMP cellsmay be grown on such membranes, added to the membrane in either anundifferentiated, partially differentiated or fully differentiated form,or ACCS or cell lysates may be added to such membranes. Alternatively,amniotic tissue in which AMP cells have not been stripped away may beused to deliver ECS cells including AMP cells to a particular site. Inall cases, ECS cells including AMP cells used in conjunction withamniotic tissue or other matrices can be used in combination with othertherapeutically useful cells and/or cells expressing biologically activetherapeutics such as those described in below.

In another embodiment, the ECS cells, including AMP cells, can be usedin combination with commercially available extracellular matrix productssuch as Oasis®, Dermagraft®, DressSkin®, Alloderm®, Promogran®, etc. Forexample, the ECS cells, including AMP cells, can be placed on top ofthese products and then applied to the wound to accelerate woundhealing. Such application may occur immediately upon combining theproducts or after incubation for a period of time.

ECS cells, including AMP cells, may be genetically engineered to producea particular therapeutic protein. Therapeutic protein includes a widerange of biologically active proteins including, but not limited to,growth factors, enzymes, hormones, cytokines, inhibitors of cytokines,blood clotting factors, peptide growth and differentiation factors.Particular differentiated cells may be engineered with a protein that isnormally expressed by the particular cell type. For example, dermalcells can be engineered to produce collagen fibers and epidermal cellscan be engineered to produce melanin.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing a nucleic acid encoding theprotein of interest linked to appropriate transcriptional/translationalcontrol signals. See, for example, the techniques described in Sambrooket al, 2001, “Molecular Cloning: A Laboratory Manual”; Ausubel, ed.,1994, “Current Protocols in Molecular Biology” Volumes I-III; Celis,ed., 1994. Details on this procedure are contained in US Publication No.2006-0222634-A1, which is incorporated herein by reference.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Preparation of AMP Cell Compositions

Recovery of AMP Cells

AMP cells were dissociated from starting amniotic membrane using thedissociation agents PXXIII, and trypsin. The average weight range of anamnion was 18-27 g. The number of cells recovered per g of amnion wasabout 10-15×10⁶ for dissociation with PXXIII and 5-8×10⁶ fordissociation with trypsin.

Culture Conditions

The primary AMP cells were cultured for 5 passages in the followingmedia: Stemline II+10% FBS, Stemline II+10% plasbumin (pb),Ultraculture+10% plasbumin (pb), and DMEM+10% FBS. Each culturecondition was tested using 15 million cells/g amnion, 10 million cells/gamnion, and 5 million cells/g amnion, depending on the enzyme used forrecovery of the primary cells. For instance, using PXXIII, 15 millioncells/g amnion were obtained, while using trypsin, 10 million cells/gamnion were obtained, while other enzymes resulted in even lesserrecovery (5 million cells/g amnion).

Passaging

Cells were passaged 5 times as follows: The cells were grown attached toa culture flask (on tissue culture treated plastic). The cells were leftto divide and grow. The cells were removed from the plastic usingTryple™ (Invitrogen), a trypsin-like product that is animal-free GMPgrade. Once unattached, the cells were centrifuged, and the cell pelletremoved and resuspended in the culture medium with protein and additives(10 ng/ml EGF) and replated back onto fresh flasks. Cells were grown ina humidified atmosphere at 37° C. and 5% CO₂.

The results indicate that the use of either Stemline or Ultraculturewith added plasbumin (pb) or albumin, the primary cultures are expandedto a level that is at least 4 fold and as much as 10 fold higher than isobtained using previous methodology (DMEM with fetal bovine serum). Eventhe use of plasbumin (pb) in the basal media DMEM resulted in anexpanded AMP cell composition, having a 3-fold increase in multipotentcells as compared to the previous method of using DMEM with fetal bovineserum. Details on these results are contained in US Publication No.2006-0222634-A1, which is incorporated herein by reference.

Another significant result observed was that cells grown in mediumcontaining plasbumin displayed a spheroidal phenotype after passaging.When the AMP cells were removed from the tissue culture surface with thedigestive enzyme and replated, AMP cells formed small clusters of cellsthat were not firmly adhered to the culture surface. Some of theclusters of cells were completely in suspension. These AMP cell clustersproliferated until up to 200 cells were present in the clusters. After aperiod of 1-5 days, the clusters of cells reattached and flattened outto form an adherent monolayer. This clustering phenotype was observed ateach passage. Further studies indicated that such clustering occurs inthe following media containing either recombinant human albumin,plasbumin, or plasmanate: OptiPRO SFM, VP-SFM, Iscove's MDM, HPGM,UltraMDCK, Stemline II and Stemline I, DMEM, and DMEM:F12, but not inAdvanced DMEM, Knockout DMEM, 293 SFM II, Pro 293S-CDM, Pro 293A-CDM orUltracultureVP-SFM.

Example 2 Use of AMP Cell and ACCS Compositions in Wound Healing

Methods.

The keratinocyte cell line isolated from epidermis (ATCC CRL-1555) wasseeded onto 6-well plates at a density of 0.3×10⁶ cells per well. Cellswere left to grow to confluency then placed into serum-free conditionsfor 48 hours. In each well a scrape or wound of the confluent monolayerwas made from the top to the bottom of the well using a 1 ml pipettetip. Images of the scrape were taken at 0, 24, 30 and 48 hours todetermine cell migration or percent of wound closure in response toaddition of ACCS to each well. Conditions tested were 0%, 50%, and 100%of the following: 1) No ACCS (control, 0%); 2) ACCS from AMP cellspassaged normally at ratio of 1:3; 3) ACCS from AMP cells that werenever passaged; 4) ACCS from AMP cells grown in the ATCC cells' media;and 5) Conditioned media from ATCC cells grown in their own media.Approximately 6 measurements were taken in microns of each scrape ateach time point using phase microscopy and MetaMorph imaging software.The percent of healing was calculated by comparing the width of eachwound at 24, 30, and 48 hours to the starting width of the wound at timezero.

Results.

ACCS from AMP cells showed a significant increase in cell migration orhealing of the scrape compared to control. CM from other cell types,however, did not show this increase. Cells that grew in ACCS from AMPcells were the only condition that showed complete closure of the scrapebefore 24 hours. ACCS from cells passaged at a ratio of 1:3 and at aconcentration of 50% (ACCS/non-CM) produced the best results. Theseresults suggest that components of ACCS from AMP cells have propertiesthat increase cell migration or wound healing.

Example 3 AMP Cells, ACCS, and Cell Lysates AccelerateRe-Epithelialization, Collagen Synthesis, and Regain to Tissue TensileStrength

The following experiment was done in an art-accepted animal model toassess whether the application of AMP cells, ACCS or AMP cell lysatescould: 1) accelerate the rate of re-epithelialization, 2) acceleratecollagen synthesis and deposition in the wound bed and 3) speed upregain to tissue tensile strength and demonstrate that transplantationof stem cells may have the same properties. It was also done to assesswhether transplanted AMP cells could incorporate into epidermal anddermal structures including follicles, glands and blood vessels.

Skin Wounding:

A pair of 6 mm diameter wounds was made on each side of the dorsalmidline. These wounds were full-thickness through the epidermis anddermis. Wounds were treated with: nothing (control), vehicle (10 mmGelfoam sponge saturated with non-conditioned media), ACCS (10 mmGelfoam sponge saturated with ACCS), hyaluronic acid vehicle (0.1 ml ofHylan A gel, Genzyme Corporation), hyaluronic acid+fluorescently (CM-DiIdye, Molecular Probes, Eugene Oreg.) labeled AMP cells (10⁶ cells/wound)or hyaluronic acid+AMP cell lysate (from 10⁶ cells/wound), immediatelyfollowing injury. The entire dorsal skin was covered with a steriledressing and secured with a biocompatible adhesive (Mastisol, FerndaleLaboratories Inc, Ferndale, Mich.). Wounds in the first three treatmentgroups were re-treated in an identical manner on days 2, 3, 4 and 5 postwounding. Following the 5th wound treatment, the wounds were leftundisturbed until day 7, at which time the Gelfoam as well as thesterile dressing was removed and the wound allowed to heal exposed tothe surrounding environment. Wounds in the last three treatment groupswere left undisturbed until time of sacrifice.

Imaging and Clinical Assessment:

Two blinded observers assessed the degree of wound healing for each ofthe 180 wound samples at the following days post injury: 1, 2, 3, 4, 5,7, 14 and 21. The following parameters were ascertained: hemostasis,wound contraction, re-epithelialization and inflammation. Digital imageswere taken of representative wound samples for each treatment group andstored for later analysis.

Tissue Analysis:

Animals were euthanized and dorsal skin was removed using aseptictechnique and each wound was individually dissected and divided. Onehalf of each wound was used for tensile strength measurements, with theother embedded for frozen sectioning and image analysis.

Tensiometry:

Wound samples from the day 7, 14 and 21 groups were analyzed bytensiometry. The results for individual specimens from one wound werecombined to determine an average TS/wound (tensile strength per wound).This value was normalized for the TS/skin (tensile strength of uninjuredskin from the opposite side); TS/wound divided by TS/skin=relativeTS/wound. The relative TS/wound was tabulated for each group at eachtime point and the mean and standard deviations determined using Exceldatabase software (Microsoft Office 2000).

Microscopic Analysis:

Tissue specimens were embedded in O.C.T. (Miles, Inc., Elkhart, Ind.)and cryostat-sectioned into approximately 10 μm thick sections, at −23°C. Thin sections, mounted on glass microscope slides, were stored inmoisture-proof slide boxes at −70° C. Representative slides wereprocessed for immunohistochemical characterization of the connectivetissue components using standard techniques. Hematoxylin and eosinstaining were used to ascertain the overall histological appearance ofthe injured mucosa. Collagen presence in the wound was assayed usingMasson's Trichrome stain. Picrosirius-polarization method was used toanalyze collagen fiber organization. Grafting and survival offluorescently labeled stem cells in the wound bed wassemi-quantitatively analyzed by measuring the total amount offluorescence present in the wound bed. Localization of cells wasrecorded and analyzed.

Effect on the rate of wound re-epithelialization and dermal collagendeposition and organization was determined. Each of these, as well asother components of the wound healing process, were analyzed usingspecific markers. Transplantation of live AMP cells into the dermalwound bed was expected to result in: 1) differentiation and engraftingof stem cells into various skin compartments and 2) continual regulatedrelease of various stem cell factors.

Results—Treatment of wounds with ACCS showed an increase in contractedgranulation formation by Day 5, and smaller wounds, greater contractionand healing by Day 14. In addition, the wounds exhibited fasterre-epithelialization and angiogenesis as compared to controls. Synthesisand deposition of collagen and regain of tissue tensile strength wereunaltered over the course of the experiment. Treatment of wounds withAMP cells showed re-epithelialization and angiogenesis at early timepoints, as well as evidence of collagen deposition and organization.Engrafted cells were not detected. No differences based on visualinspection in clinical observations (redness, swelling, size, etc.) wereseen nor was regain of tissue tensile strength altered over the courseof the experiment.

Example 4 Detection of Cytokines in ACCS and Unconditioned Media Samples

In addition to multipotency, AMP cells may play a significant role inthe inflammatory response. In the early phases of wound healing,chemokines and cytokines regulate chemotaxis and activation ofinflammatory cells. Growth factors play dominant roles in regulatingcell proliferation, differentiation, and synthesis of extracellularmatrix. Amnion epithelial cells have been shown to secrete manycytokines and growth factors. These factors include prostaglandin E,PDGF, TGF-α, EGF, IL-4, IL-8, TNF, interferons, activin A, noggin,b-FGF, angiogenic factors, and other neuroprotective factors (Koyano,S., et al., (2002) Dev Growth Differ 44, 103-12; Blumenstein, M., etal., (2000) Placenta 21, 210-7; Tahara, M., et al., (1995) J ClinEndocrinol Metab 80, 138-46; Paradowska, E., et al., (1997) Placenta 18,441-6; Denison, F. C., et al., (1998) Hum Reprod 13, 3560-5; Keelan, J.A., (1998) Placenta 19, 429-34; Sun, K., et al., (2003) J ClinEndocrinol Metab 88, 5564-71; Uchida, S., et al., (2000) J Neurosci Res62, 585-90).

Many of these cytokines are associated with wound healing and some havebeen credited with contributing to scarless healing in the fetus(Robson, M. C., et al., (2001) Curr Probl Surg 38, 72-140; Ferguson, M.W. et al., (2004). Philos Trans R Soc Lond B Biol Sci 359, 839-50).

To determine which of these cytokines may be secreted by the AMP cellsof the present invention, ACCS was isolated from cell cultures that wereseeded onto tissue culture treated flasks at a density of ˜40,000 cellsper cm². Cells were cultured in a proprietary serum-free mediumsupplemented with 10 ng/ml of EGF. Culture media was exchanged every 2days during the growth period. After cells reached near confluency (˜1-2wk after isolation), fresh media was applied and ACCS was collectedafter three days and stored at −80° C. for subsequent analysis.

ACCS was analyzed for secreted protein content via antibody arrays formultiple protein detection (RayBiotech, Norcross, Ga. using RayBio®Human Cytokine Antibody Arrays V, VI, and VII). The samples that wereanalyzed were complete unconditioned media+plasbumin; completeUnconditioned media+EGF (no plasbumin); ACCS from placenta 1+plasbumin;ACCS from placenta 1 (no plasbumin); and ACCS from placenta 2+plasbumin.

Results—The following wound healing relevant cytokines were detected inACCS by immunoblot: Angiopoietin-2, Angiogenin, bFGF, EGF, FGF-7, FGF-4,IGF-1, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, PDGF-AA, PDGF-AB,PDGF-BB, PDGF-Ra, PDGF-Rb. The following wound healing relevantcytokines were not detected in ACCS by immunoblot: TGFα, TGFβ1, TGFβ2,TGFβ3.

Example 5 Detection of Cytokines in Non-Pooled and Pooled ACCS UsingELISA

ELISAs were performed on non-pooled and pooled ACCS because it is a moresensitive assay and because the results are quantitative. ELISAs wereperformed on conditioned media (ACCS) derived from AMP cells obtainedfrom 10 different placentas (non-pooled ACCS). In addition to assayingeach ACCS sample individually, pooled ACCS samples were also tested todetermine if variability of ELISA results between samples could bereduced. ACCS was obtained as follows: AMP cells were isolated from theamnion as described in Example 1 above, seeded at 10×10⁶ cells/10 mlmedia/T75 tissue culture flask and cultured until confluent. Onceconfluent, the media was changed and ACCS was collected 3 dayspost-confluence. ACCS was centrifuged to remove any cellular debris,aliquoted, and stored at −80° C. Pool 1 was comprised of ACCS fromplacentas 1-5, Pool 2 was comprised of ACCS from placentas 6-10, andPool 3 was comprised of ACCS from placentas 1-10.

Results: In the non-pooled ACCS samples, PDGF levels ranged from about75-165 pg/ml whereas in Pool 1, 2 and 3 the levels ranged from about110-170 pg/ml. In the non-pooled ACCS samples, Angiogenin levels rangedfrom about 3.5-4.5 ng/ml whereas in Pool 1, 2 and 3 the levels rangedfrom about 3.5-4.5 ng/ml. In the non-pooled ACCS samples, VEGF levelsranged from about 4-18 ng/ml whereas in Pool 1, 2 and 3 the levelsranged from about 6-16 ng/ml. VEGF was not detected in the lesssensitive immunoblot assay described in Example 4 above. In thenon-pooled ACCS samples, TGFβ2 levels ranged from about 1-4.5 ng/mlwhereas in Pool 1, 2 and 3 the levels ranged from about 2.5-2.7 ng/ml.TGFβ2 was not detected in the less sensitive immunoblot assay describedin Example 4 above. In the non-pooled ACCS samples, TIMP-1 was 0.68μg/ml (as measured by a multiplex assay). In the non-pooled ACCSsamples, TIMP-2 level was 1.05 μg/ml. Pooling of ACCS had the effect ofreducing variability of secreted factor levels as compared to thosemeasured in the non-pooled ACCS samples. This creates a more consistentproduct that contains physiological levels of cytokines and growthfactors similar to those reported in the literature for acute woundhealing. It also provides an efficient method of creating the moreconsistent product, thus reducing manufacturing costs.

Example 6 AMP Cell/Fibroblast Co-Cultures to Create Amnion-DerivedMulticellular Dressings (AMDs)

Co-Cultures

It has been reported in the literature that under certain conditionswhen ES cells are co-cultured with fibroblasts, the ES cells are inducedto differentiate into keratinocyte-like cells. To determine what effectco-culture of AMP cells with fibroblasts would have on AMP cells, anexperiment was done in which 3.3×10⁶ AMP cells were co-cultured with0.4×10⁶ fibroblasts on a collagen IV-coated T25 flask for 3, 5, 10, 15,and 25 days.

Results:

When treated with the trypsin-like enzyme Tryple (Invitrogen), both AMPcell cultures and fibroblast cell cultures alone release cells as asingle cell suspension. However, when the AMP cell/fibroblast co-culturewas treated with Tryple, the cells came off the treated culture surfaceas sheets rather than as a single cell suspension. Furthermore, thesheets were very stable and somewhat resistant to enzymatic andmechanical disruption.

It is theorized that these sheets may be suitable for use as wounddressings when it is desirable to have a dermal-type graft. Withdemonstrated recent success with mitral resuscitation, management ofinhalation injuries, control of burn wound sepsis, and understanding ofthe hypermetabolic response, early excision and rapid closure of theburn wound with a serviceable integument becomes a therapeuticimperative. In small surface area burns, this can be accomplished byautogenous skin grafts. For large surface area burns, both partial andfull-thickness, there is not yet a totally satisfactory solution.Cutaneous epithelial autografts can be grown from the patient's skin andmassively expanded to cover the entire body. Unfortunately, the lack ofdermis leads to prolonged fragility and significant scarring, therefore,many believe that a “dermis” is required along with an epithelium.

Recent products with a supposed dermal substitute or neodermis such asIntegra, Alloderm, Transcyte, Apligraf, and Dermagraft have attemptedsolve the problem. However, all of these “skin” substitutes have theproblem of being expensive and having lower resistance to infection thanautografts. Without a satisfactory rapid reliable wound closure for burninjuries, the wound remains in the inflammatory phase of healing for aprolonged period of time resulting in excessive scarring.

Robson et. al., (Robson, M. C., and Krizek, T. J. (1973) Ann Surg 177,144-9.) reported success in treatment of experimental and clinical burns(both partial and full thickness) using human amniotic membranes. It wasthought that part of the effect seen from the treatment with amnioticmembranes was due to a humoral substance or substances stimulating woundhealing. These observations were prior to present knowledge of cytokinesand growth factors. More recently, attempts have been made to userecombinant growth factors and growth hormones to affect more rapidhealing of the burn wound. Amniotic membranes proved not to be practicalbecause of the risk of virally transmitted diseases. However, theobservations from those early experiments and coupled with new knowledgesupport the possibility that the multipotentiality of AMP cells andtheir now demonstrated and described herein protein secretory profile ofcytokines and other humoral substances stimulatory for wound healing maybe useful in providing rapid early closure for thermal injuries.

Co-Culture of AMP Cells/Fibroblasts on ECM to Create Amnion-DerivedMulticellular Dressings (AMDs):

AMP cells were collected at TO and seeded onto coated T25's tissueculture flasks at a density of 3.3×10⁶. AMP cells were allowed to attachand begin to proliferate (1-2 days). Human foreskin fibroblasts wereseeded into the same flask at a density of 0.2×10⁶ (this density may bevaried). Co-cultures were supplemented every other day with Celprogenstem cell keratinocyte differentiation media (other suitable mediainclude but are not limit to EpiLife and UltraCulture). Co-cultures canbe maintained up to 30 days or more. Transformation into removable‘sheet’ of cells usually occurs around day 14 and can be assessed byobserving cell morphology. The ‘sheet’ of cells was removed byincubation in Tryple (Invitrogen). The ‘sheet’ of cells was then placedon Oasis, a commercially available ECM (other suitable ECMs include butare not limited to Alloderm and DressSkin), and allowed to grow indifferentiation conditions until an AMD formed, then fixed, andsectioned. The AMD was then assayed by IHC for differentiation markersand HE for organization and morphology.

Results: The results of this experiment demonstrated that AMP cells growand proliferate to form a stratified phenotype on Oasis and Alloderm asdetermined by HE.

The above experiment was also be done with AMP cells collected at theend of p0 after having been primed in differentiation conditions beforeseeding on Oasis. The results of this experiment demonstrated that thecells attached to the Oasis and exhibited a stratified phenotype.

Example 7 Effects of ACCS in an Animal Model of Acute Wound Healing

An art-accepted animal model of acute excisional granulating wound wasused to evaluate the effect of ACCS on wound healing. Details arecontained in US Publication No. 2006-0222634-A1, which is incorporatedherein by reference. The animals were divided into the following groups:Group I—ACCS, non-infected; Group II—Unconditioned media; GroupIII—ACCS, infected; Group IV—Unconditioned media, infected.

Analog tracings were made every 72 hours onto acetate sheets of bothopen wound areas and of the advancing full-thickness skin edges of allwounds. To eliminate site-related variability in the wounds, only thethree caudal wounds were measured for statistical purposes, since themost cephalad wound has been shown to demonstrate different healingcharacteristics. Wound area calculations were performed with the use ofdigital planimetry (Sigma Scan; Jandel Scientific, Corte Modera,Calif.). Weekly quantitative bacterial analyses were performed on asubset of wounds in each group and are expressed as CFUs/g of tissue.

After all four wounds of each animal were completely epithelialized asdetermined by visual inspection, the animals were euthanized and theentire dorsum of the rat including the panniculus carnosus was removed.A 1 cm wide skin strip perpendicular to each resultant scar, washarvested for breaking strength analysis. An Instron tensiometer (ModelNo. 4201; Instron Corp., Canton, Mass.) with a 5 kg tension load celland cross head speed of 10 mm/min was used. Breaking strength is definedas the force required to rupture the scar and is reported in kilograms.

Results

The application of ACCS overcomes the inhibition of wound healing causedby bacteria and shifts the healing trajectory in contaminated wounds tothat of near normal healing.

Example 8 Effects of ACCS in an Animal Model of Chronic Wound Healing

An art-accepted animal model for chronic granulating wound was used tostudy the effects of ACCS on chronic wound healing (Hayward P G, RobsonM C: Animal models of wound contraction. In Barbul A, et al: Clinicaland Experimental Approaches to Dermal and Epidermal Repair: Normal andChronic Wounds. John Wiley & Sons, New York, 1990).

Results: ACCS was effective in not allowing proliferation of tissuebacterial bioburden. ACCS allowed accelerated healing of the granulatingwound significantly faster than the non-treated infected control groups.

Example 9 Characterization of AMP Cells at p0

Method of obtaining selected AMP cells: Cells were plated immediatelyupon isolation from the amnion. After ˜2 days in culture non-adherentcells were removed and the adherent cells were kept. This attachment toplastic tissue culture vessel is the selection method used to obtain thedesired population of cells. Adherent and non-adherent AMP cells appearto have a similar cell surface marker expression profile but theadherent cells have greater viability and are the desired population ofcells. Adherent AMP cells were cultured until they reached˜120,000-150,000 cells/cm². At this point, the cultures were confluent.Suitable cell cultures will reach this number of cells between ˜5-14days. Attaining this criterion is an indicator of the proliferativepotential of the AMP cells and cells that do not achieve this criterionare not selected for further analysis and use. Once the AMP cells reach˜120,000-150,000 cells/cm², they were collected and cryopreserved. Thiscollection time point is called p0 and all subsequent analyses are donewith thawed p0 AMP cells.

Analysis of thawed p0 AMP cells: The AMP cells were analyzed for theexpression of several cell surface markers. Table 1 below shows theresults of this cell surface maker analysis as well as the % positive ofthese same markers upon isolation of the cells from the amnion. As canbe seen, with the exception of HLA-G (whose expression goes down) andCD90 (whose expression goes up), the other tested cell surface markersremain constant over time.

TABLE 1 % positive at % positive at Cell Surface Marker isolation p0CD90 >95 >95 CD29 >95 >95 SSEA4 70-90 70-90 CD10 70-90 70-90 CD44 <1 <1CD45 <1 <1 HLA-G (MEMG/9 ab) >60 10-50 CD90 <1 10-50

Example 10 Generation of ACCS

The AMP cells of the invention can be used to generate ACCS. The AMPcells were isolated as described herein and 10×10⁶ cells were seededinto T75 flasks containing 10 ml culture medium. The cells are cultureduntil confluent, the medium is changed and ACCS was collected 3 dayspost-confluence. Skilled artisans will recognize that other embodimentsfor collecting ACCS from confluent cultures, such as using other tissueculture vessels, including but not limited to cell factories, flasks,hollow fibers, or suspension culture apparatus, are also contemplated bythe methods of the invention. It is also contemplated by the instantinvention that the ACCS be cryopreserved following collection.

Example 11 Effects of AMP Cells and ACCS in Two Animal Models of WoundHealing

The two art-accepted animal models of granulating wounds described abovewere used to evaluate the effect of AMP cells and ACCS on wound healing.The experimental groups for these experiments are Group1-Non-contaminated control (UCM only); Group II—Contaminated, ACCS;Group III Contaminated, AMP cells; Group IV—Contaminated AMP cells+ACCS;and Group V—Contaminated control.

Results: Neither ACCS, AMP cells, or AMPs cells+ACCS had any effect onbacterial load in the acute contaminated wound model at either Day 0 orDay 8, demonstrating that none of the treatments are antimicrobial.However, ACCS (II), AMP cells (III) and AMP cells+ACCS (IV) were allable to accelerated wound healing as compared to contaminated control(V) and UCM (I). The finding that all treatment groups can shift thehealing curve to the left even when the wound is infected represents asignificant improvement over currently available treatments.

The acute contaminated wounds were tested for breaking strength on Day22 (Robson, et al: The effect of cytokine growth factors on theprevention of acute wound failure. Wound Rep Regen 12: 38-43, 2004.Franz, et al: Fascial incisions heal faster than skin. A new model forabdominal wall repair. Surgery 129: 203-208, 2001). In the AMP cell andAMP cell+ACCS treated groups, there was a statistically significant(=p<0.05) increase in breaking strength when the results of twoexperiments were added together (13.5N each) as compared to UCM (10.5N)and ACCS alone (10N). As Day 22 is late in the wound healing timecourse, it is hypothesized that if breaking strength is tested earlierin the wound healing process, the difference between treated anduntreated will be even greater.

Similar experiments as those described above for an acute contaminatedwound model were performed using a chronic contaminated wound model(Hayward P G, Robson M C: Animal models of wound contraction. In BarbulA, et al: Clinical and Experimental Approaches to Dermal and EpidermalRepair Normal and Chronic Wounds. John Wiley & Sons, New York, 1990).Neither ACCS, AMP cells, nor AMP cells+ACCS had a significant impact onbacterial load at any of the days tested (Days 0, 8, 10), demonstratingagain that none of the treatments are antimicrobial. Furthermore, ACCS(II), AMP cell (III), AMP cells+ACCS (IV) are able to accelerate woundhealing significantly as compared to UCM (I) and contaminated control(VI). To achieve a 50% open wound, contaminated control took 18 days,whereas ACCS, AMP cells and ACCS+AMP cells achieved this by day aboutday 10-11. This shift of 6 days represents a significant improvement inhealing rates than those achievable by currently available therapies.

Example 12 Evaluation of Accelerated Wound Strength And Prevention ofAcute Wound Failure

One object of the invention is to decrease wound failure in surgical andtraumatic injuries by treating these acute wounds with ACCS from AMPcells. The focus is muscle, fascial and skin wound healing in vivofollowing surgical injury. Wound fibroblasts are isolated to measure theeffect of soluble mediators derived from AMP cells on repair fibroblastfunction in vitro.

Art-accepted animal models for evaluating wound strength and woundfailure (Robson, et al: The effect of cytokine growth factors on theprevention of acute wound failure. Wound Rep Regen 12: 38-43, 2004.Franz, et al: Fascial incisions heal faster than skin. A new model forabdominal wall repair. Surgery 129: 203-208, 2001) were used inexperiments to assess whether or not ACCS could increased wound strengthand decrease wound failure.

The animals were randomly assigned into one of 12 Groups. InExperimental Designs 1 and 2, each of the three animal models (Shamlaparotomy, Healing laparotomy and Hernia) were treated with fourexperimental conditions of ACCS containing the humoral products of AMPcells. (No treatment, Control AMP cell media (0% conditioned), 50% ACCSand 100% ACCS). 100 IU of media is delivered to the site of thelaparotomy myofascial and skin incisions prior to wounding.

Results: The application of ACCS resulted in a statistically significantincrease in breaking strength (=p<0.05) (16.7N) at Day 7 as compared toPBS (8N) and UCM (10.5N) in this animal model of wound failure. Inaddition, in this same animal model, the addition of ACCS resulted in astatistically significant increase in tensile strength (=p<0.05)(0.34N/mm²) at Day 7 as compared to PBS (0.21N/mm²) and UCM (0.23N/mm²).These data indicate that ACCS is capable of increasing wound strength inthis model.

Results obtained in the incisional hernia model: In the treatment groupin which the incisions were “primed” with 100 μl ACCS only 25% of theanimals formed incisional hernias, as compared to PBS or UCM (controlgroups) in which 100% of the animals developed incisional hernias.Furthermore, when the hernias were removed and their size measured, theACCS treated group had an average hernia size that was ⅛ the size of thePBS treated controls and 1/12 the size of UCM controls. In addition, Inthe ACCS treated incision, there is no visible indication of theincisional wound, whereas in the UCM treated incision, there is anobvious visible suture line. A histological section through the surgicalsite in both an UCM treated and the ACCS treated animal reveals that inthe ACCS treated specimen, there is thick, organized fascia (F), anorganized and intact rectus muscle (RM) and a well healed peritoneum(P). These features are not evident in the UCM treated specimen. Takentogether, these data clearly demonstrate that the application of ACCSprior to incisional injury can increase wound strength, decrease orprevent wound failure as evidenced by both reduced rate of herniaformation and hernia size, and accelerate wound healing.

Histology: Histological analyses of provisional matrix structure,fibroblast migration, inflammatory response and wound angiogenesis isused to compare the groups using H&E and trichrome staining of samplesare collected from laparotomy wounds or incisional hernias from rats.The density of wound collagen formation is measured using antibodiesspecific for rat collagen types I and III (Chemicon International, Inc.,Temecula, Calif.). Cellular infiltration into the wounds at eachtime-point is measured as the mean cell number from three high-poweredfields by a blinded observer using a microscope. In addition,histological specimens are digitized using a UMAX Astra 1200S scannerand analyzed using the computer software application Adobe PhotoShopversion 5.0. Differences in cellularity and intensity of collagenstaining are compared using the Students t test (SigmaStat, Jandel).

Example 13 Use of AMP Cells, ACCS, Cell Lysates, and Cell Products forRapid Early Wound Closure of Thermal Injuries

Outcome and rehabilitation of thermal injuries rely on early burn woundexcision and rapid wound closure. The speed of wound closure with aserviceable integument or integument substitute is the key to animprovement in survival. Providing novel approaches that will facilitateearly, rapid wound closure, while minimizing long-term scarring, is anobject of the present invention.

Established in vivo animal models were used to evaluate the use of AMPcells and ACCS for early, rapid wound closure of partial-thickness andfull-thickness burns.

It is theorized that AMP cells can differentiate into mesodermal andectodermal cells. Thus, it may be possible that use of such cells willprovide early and permanent closure of the burn wound. Since presently,the prolonged time the wound is in the inflammatory phase is the knownvariable leading to proliferative scarring, it is expected that early,permanent closure of the burn wound would result in decreased scarringand, thus, increased function.

Three animal models of partial-thickness and full-thickness thermalinjuries were used (DelBecarro, et al: The use of specific thromboxaneinhibitors to preserve the dermal microcirculation after burning.Surgery 87: 137-141, 1980. Robson, et al: Increasing dermal perfusionafter burning by decreasing thromboxane production. J Trauma 20:722-725, 1980. Polo, et al: An in vivo model of human proliferativescar. J Surg Res 74: 187-195, 1998). The three models are differentbecause the first mimics partial-thickness healing by epithelializationin approximately three weeks while the second and third mimicfull-thickness healing by contraction and epithelialization and canremain unhealed for up to eight weeks. The difference in the last twofull-thickness wounds is the host. One group is a normal rat with anintact immune system, while the other is an athymic “nude” rat which isdevoid of T-lymphocytes.

Partial-thickness wound results: Healing of partial-thickness (seconddegree) scald burns in guinea pigs is was accelerated using topicalapplication of ACCS and/or topical application of AMP cells. CombiningACCS and AMP cells resulted in the greatest acceleration of burn woundhealing.

Full thickness wound results: The rate of wound healing was acceleratedin all of the treated groups (III, IV, and V) as compared to the twocontrol groups (no treatment group (I) and UCM treated group (II)).

Example 14 Bone Differentiation Assays with AMP Cells

This initial series of alkaline-phosphatase (ALP) activity stainingsuggests differentiation of AMP cells towards an osteogenic lineageunder the influence of both BMP2 and osteogenic supplement (OS) media.The experiment began at passage three, with AMP cells seeded below theideal density. In general, cells cultured in osteogenic supplement (OS)assumed a flattened morphology and spread to create confluent layers onthe flask surface.

Positive staining for ALP activity was observed as early as day three inAMP cells cultured in OS media, independent of BMP2 concentration. NoALP activity was observed in cultures supplemented only with BMP2 withinthree days. By day seven, the number of cells expressing ALP increasedin all cultures under OS. Under OS and at high (100-200 ng/ml) BMP2doses, the intensity of ALP staining declined indicating down-regulationof ALP at this time (expected). By day seven, ALP activity was seen incells cultured in basal AMP cell media supplemented with BMP2. Inparticular, a large number of cells supplemented only with 200 ng/mlBMP2 showed strong ALP activity at day seven. By day ten, ALP expressionwas no longer evident in populations cultured with both OS mediasupplemented with higher (50-200 ng/ml) concentrations of BMP2.Presumably, ALP activity has decreased as the cells progress towardsosteogenic lineage (this is the expected outcome). ALP staining canstill be seen in OS media with lower concentrations of BMP2. At day ten,cells cultured without OS appear unhealthy and sparse, for reasons thatare not clear. These data indicate that AMP cells cultured in anosteogenic supplement media exhibit markers associated with osteogenicdifferentiation.

Example 15 Ability of AMP Cells to Promote Complete Regeneration of DeepWounds

Experiments are designed to promote complete regeneration of deep woundsthrough re-creating the all of the necessary tissues including bone,muscle, cartilage, skin, and neural tissue. Initially, in vitroexperiments are designed to determine if AMP cells can differentiateinto all of the cells of interest. AMP cells will be cultured aspreviously described. Mesenchymal stem cells (Cambrex, Rutherford, N.J.)will be used as a control for differentiation experiments. MSC's will beseeded at 5,000-6,000 cells per cm² and cultured in Mesenchymal StemCell Growth Medium (MSGM, Cambrex, Rutherford, N.J.).

Osteogenic:

Once cells are confluent, growth media will be changed (DMEM, 10% FBS,1% pen/strep) to osteogenic differentiation media (Shi, Y. Y., et al.,(2005) Plast Reconstr Surg 116, 1686-96.) (DMEM, 10% FBS, 1% pen/strep,250 uM ascorbate-2-phosphate, 10 mM beta-glycerophosphate, 2.5 uMretinoic acid). Osteogenic differentiation media will be changed every2-3 days. Alkaline phosphatase activity of adipose-derived mesenchymalcells will be evaluated in duplicate wells after 7 days of culture.Alkaline phosphatase staining will be performed using the AlkalinePhosphatase Staining Kit (Sigma) following the manufacturer'srecommendations. Experiments will be performed in triplicate. Von Kossastaining will be performed in duplicate wells to assess the ability ofcells to mineralize the extracellular matrix and form bone nodules.Staining will be performed on cells after 21 days of culture induplicate wells in differentiation media conditions. Cells will be fixedin neutral buffered formalin for 30 minutes, incubated with 1% aqueoussilver nitrate for 15 minutes under ultraviolet light, stained with 5%sodium thiosulfate for 2 minutes, and finally counterstained with 1%Safranin 0 for 10 minutes. In addition, calcium concentration in theextracellular matrix will be determined via a biochemical colorimetricassay using the Calcium Reagent Set (Biotron Diagnostics, Hemet, Calif.)in duplicate wells. Experiments will be performed in triplicate.

Adipogenic:

AMP cells and MSC will be cultured in adipogenic differentiation media(Shi, Y. Y., et al., (2005) Plast Reconstr Surg 116, 1686-96.) for 3days (DMEM, 10% FBS, 1% pen/strep, 10 ug/ml insulin, 1 uM dexamethasone,0.5 mM methylxanthine, 200 uM indomethacin), then change to adipocytemaintenance media for 2 more days (DMEM, 10% FBS, 1% pen/strep, 1 ug/mlinsulin). Oil Red O staining will be performed to assess for adipogenicdifferentiation in duplicate wells (as indicated by the presence ofintracellular lipid-filled droplets) after 5 days of culture inadipogenic media. Cells will be fixed in 10% neutral buffered formalinfor 30 minutes and then incubated in 60% Oil Red O solution for 30minutes at 37° C. Experiments will be performed in triplicate.

Chondrogenic:

AMP cells and MSC will be cultured in standard non differentiationconditions and then collected and resuspended at 1×10⁷ cells/mlconcentration. Ten μl droplets will then be placed onto a culture dishand allowed to adhere to substratum at 37° C. for 2 hours. Thenchondrogenic media (Malladi, P., et al., (2006) Am J Physiol CellPhysiol 290, C1139-46.) will be added carefully around cell aggregates(DMEM, 1% FBS, 1% pen/strep, 37.5 ug/ml ascorbate-2-phosphate, ITSpremix (BD Biosciences), 10 ng/ml TGF-B1 (Research Diagnostics, Inc.,Flanders, N.J.). Micromasses will be fixed in 4% paraformaldehyde with4% sucrose for 15 minutes, embedded with Optimal Cutting Temperature(O.C.T.) compound. Ten gm cryosections will be mounted on slides andstained by hematoxylin and eosin and alcain blue. Immunohistochemistrywill be performed as follows. Sections will be blocked at roomtemperature for 30 minutes and incubated with primary antibody at 4° C.overnight (anti-collagen II, Santa Cruz Biotechnology, Santa Cruz,Calif.). Followed by secondary antibody (Vector Labs, Burlingame,Calif.) incubation, 8 sections will be labeled with ABC reagent (VectorLabs, Burlingame, Calif.) for 10 minutes at room temperature. DAB(Vector Labs, Burlingame, Calif.) was applied to each section andhematoxylin will be used for counterstaining.

Skeletal Myogenic:

AMP cells and MSC will be cultured as previously described. Skeletalmyogenic differentiation will be induced by culturing cells in myogenicmedium (Gang, E. J., et al., (2004) Stem Cells 22, 617-24.) (culturemedium supplemented with 5% horse serum, 0.1 μM dexamethasone, and 50 μMhydrocortisone) for up to 6 weeks. Myogenic differentiation willanalyzed by FACS for MyoD1, myogenin, and myosin heavy chain (MyHC). ForFACS, cells will be detached and stained sequentially with primaryantibodies (human-anti MyoD and anti-myogenin antibodies; BectonDickinson) and FITC-conjugated secondary antibodies (FITC-rat anti-humanIgG1; Becton Dickinson). Cells will be fixed with 2% formaldehyde untilanalysis with FACS. For detection of an intracellular protein MyHC,cells were permeabilized with cold methanol/PBS for 2 minutes at −20° C.before staining with primary mouse anti-myosin (fast, Sigma) andFITC-conjugated secondary antibody.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Throughout the specification various publications have been referred to.It is intended that each publication be incorporated by reference in itsentirety into this specification.

1.-17. (canceled)
 18. A method for accelerating wound healing of achronic, infected wound as compared to the healing rate of a non-treatedchronic, infected wound in a patient in need thereof comprising treatingthe chronic, infected wound by topically administering to the patient'schronic, infected wound 0.5 μL-2000 μL conditioned medium per cm² ofchronic, infected wound, wherein the conditioned medium is made by a)obtaining a placenta and isolating an amnion from the placenta; b)enzymatically releasing amnion-derived epithelial cells from the amnion;c) collecting the released amnion-derived epithelial cells; d) culturingthe collected amnion-derived epithelial cells of step (c) in basalculture medium that is supplemented with human serum albumin and humanrecombinant EGF; and e) collecting the conditioned medium.
 19. Themethod of claim 18 wherein the chronic, infected wound is selected fromthe group consisting of a pressure ulcer, a venous ulcer, a diabeticulcer and a sickle cell ulcer.
 20. The method of claim 18 wherein theconditioned medium is pooled conditioned medium.
 21. The method of claim18 wherein the conditioned medium is administered in combination with anactive agent selected from the group consisting of a growth factor, acytokine, a chemokine, an antibody, an antibiotic, an anti-fungal, andan anti-viral.